RAPID COMMUNICATION

Quantitation of AgNORs by Flow Versus Image Cytometry

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

	Summary

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

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. (J Histochem Cytochem 49:433–437, 2001)

Key Words: AgNORs, flow cytometry, image cytometry, cell cycle time, growth fraction, cell proliferation, tumor prognosis

	Introduction

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

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 (Goodpasture and Bloom 1975 ). It was established in vitro that the AgNORs are as large as the cell doubling time is short (Trere et al. 1989 ; Derenzini et al. 1990 ; Ofner et al. 1992 ). These results were confirmed in vivo because the doubling time of human cancer xenograft tumor mass in athymic mice was reported to increase as the AgNOR quantity decreased (Trere et al. 1997 ). Finally, experimental modulation of the cell cycle duration by temperature showed that the AgNOR size decreased as the cell cycle lengthened (Canet et al. 2001), strongly suggesting that the AgNOR size is a marker of the cell cycle speed. In oncology, Ploton et al. 1986 demonstrated that human prostate cancer cells are characterized by a higher number of interphase AgNORs compared to corresponding benign or hyperplastic cells. Since that time, many investigations have reported a significant prognostic value of AgNOR area for cancer outcome in 80 of 104 cases examined (Pich et al. 2000 ).

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) (Trere 2000 ). However, counting proved to be poorly reproducible (Trere et al. 1995 ) and imaging suffered from the limited number of analyzed cells and poor interlaboratory reproducibility. Moreover, the tumor-to-tumor comparison of the AgNOR size should concern only the cycling cells, as emphasized by Bigras et al. 1996 , thus making it necessary to use an additional marker for the cells in cycle, such as the KI-67/MIB1 antigen revealed by immunochemistry.

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.

	Materials and Methods

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

To investigate the effect of silver precipitates on the scattered light, cells were treated by the usual procedure described by Ploton et al. 1986 with (treated cells) or without (control cells) silver salts. The effect of silver precipitates was measured as the difference of the intensity of the forward scattered light between treated and control cells. To compare results obtained by FCM and results obtained by ICM and to verify the usual correlation between AgNOR area and cell cycle time, the experiments were performed on three different mammary cell lines (MCF-7, T47-D, and ZR 75-30) having different doubling times, which were 29.8 hr, 38 hr, and 150 hr, respectively. MCF-7 and T47-D cell lines were cultured in DMEM medium complemented with 10% fetal calf serum (FCS), L-glutamine (200 mM), streptomycin/penicillin (250 U/ml), amphotericin (150 U/ml), insulin (0.2 U/ml), and 1% non-essential amino acids. The ZR 75-30 cell line was cultured in RPMI 1640 complemented with 10% FCS and 1 mM sodium pyruvate. All cultures were performed in a humidified 5% CO₂ incubator at 37C. All products were supplied by Life Technologies (Cergy Pontoise, France).

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 GAM–alkaline 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 GAM–APase. 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 PBS–5% sodium thiosulfate. For controls, fixed cells were suspended directly in PBS–5% sodium thiosulfate.

For ICM analysis, cell fixation and staining were carried out as described by Canet et al. in press . Conditions for fixation and silver staining were exactly the same as those for cell suspension described above.

FCM was performed using a FACscan system (Becton–Dickinson; San Jose, CA) managed by CellQuest software (Becton–Dickinson). 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.

	Results

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

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.

View larger version (9K): [in this window] [in a new window]   Figure 1. Effect of silver staining in the forward scattered light using the T47-D cell line. (A) FSC profile of unstained control (solid histogram) and silver-stained (outlined histogram) cell populations. (B) FSC against SSC dot-blots of unstained control cells and silver-stained cells.

View larger version (14K): [in this window] [in a new window]   Figure 2. Comparison between the effect of silver staining in the FSC and the AgNOR relative area measured by ICM with the corresponding standard deviations.

View larger version (73K): [in this window] [in a new window]   Figure 3. Example of MCF-7 (A) and ZR 75-30 (B) Ki-67/AgNOR double-stained cells and their relative AgNOR area (RA) measured by ICM. Arrows point to one silver dot for each cell line.

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).

View larger version (12K): [in this window] [in a new window]   Figure 4. Correlation between effect of silver staining on the FSC and the cell doubling time with the corresponding SD.

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 (Gerdes et al. 1983 ). As shown Fig 5A, the red fluorescence of the Ki-67–MIB1–APase–Fast Red precipitate was detected in the FL3 channel, which detects wavelengths above 600 nm. Those cells with a fluorescence intensity lower or equal to that of the APase control cells were considered negative for Ki-67/MIB1. Interestingly, the APase–Fast Red precipitate did not interfere with the FSC measurement of unstained control cells because their respective histograms showed an obvious registration (Fig 5B).

View larger version (11K): [in this window] [in a new window]   Figure 5. Ki-67/MIB1 staining with the APase–Fast Red procedure. (A) Ki-67/MIB1 fluorescence profile of stained cells (solid histogram) and unstained control cells (outlined histogram). (B) FSC of Ki-67/MIB1-stained cells (solid histogram) and unstained control cells (outlined histogram).

	Discussion

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

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. Mitsui 1992 reported that staining leukemia cells with silver nitrate at low concentration increased the SSC. This parameter was not selected in this study because it proved to be unstable (data not shown) and was known to be dependent on the shape and orientation of the scattering particles, unlike FSC.

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 (Pich et al. 2000 ). Much better correlations were obtained when AgNOR measurement concerned only the cycling cells, usually identified by marking with the Ki-67/MIB 1 antigen (Bigras et al. 1996 ; Lorenzato et al. 2000 ). Measuring both the fraction of cells in cycle (G) and the duration of the division cycle (T) is known to provide a complete and comprehensive assessment of proliferative activity in tumors defined as G*1/T (van Diest et al. 1998 ). Until the present, quantitation of AgNORs has been performed exclusively by ICM, which is time-consuming, limited to a small fraction of the tumors, and poorly reproducible among laboratories using different imaging setups, and is therefore not in routine use. The FCM procedure reported here is a good candidate to support a standardized assessment of the cell proliferation status of tumors.

Received for publication November 9, 2000; accepted December 6, 2000.

	Literature Cited

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

Bigras G, Marcelpoil R, Brambilla E, Brugal G (1996) Interest of targeting AgNOR measurement in cycling cells: in vivo cell kinetic evaluation of non-small cell lung cancer. Anal Cell Pathol 11:183-198[Medline]

Canet V, Montmasson MP, Giroud F, Brugal G (in press) Correlation between AgNORs area and cell cycle time. Cytometry

Derenzini M, Pession A, Trerè D (1990) The quantity of nucleolar silver-stained proteins is related to proliferating activity in cancer cells. Lab Invest 63:137-140[Medline]

Derenzini M, Ploton D (1991) Interphase nucleolar organizer regions in cancer cells. Int Rev Exp Pathol 32:150-192

Gerdes J, Schwab U, Lemke H, Stein H (1983) Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer 31:13-20[Medline]

Goodpasture C, Bloom SE (1975) Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining. Chromosoma 20:37-50

Lorenzato M, Abboud P, Lechki C, Browarnyj F, O'Donohue MF, Ploton D, Adnet JJ (2000) Proliferation assessment in breast cancer: a double-staining technique for AgNOR quantification in MIB-1 positive cells especially adapted for image cytometry. Micron 31:151-159

Mitsui H (1992) AgNORs. Nippon Rinsho 50:2349-2354[Medline]

Ofner D, Hittmair A, Marth C, Ofner D, Totsch M, Daxenbichler G, Mikuz G, Margreiter R, Schmid KW (1992) Relationship between quantity of silver-stained nucleolar organizer regions associated proteins (Ag-NORs) and population doubling time in ten breast cancer cell lines. Pathol Res Pract 188:742-746[Medline]

Pich A, Chiusa L, Margaria E (2000) Prognostic relevance of AgNORs in tumor pathology. Micron 31:133-141

Ploton D, Menager M, Jeannesson P, Himber G, Pigeon F, Adnet JJ (1986) Improvement in the staining and in the visualization of the argyrophilic proteins of the nucleolar organizer regions at the optical level. Histochem J 8:5-14

Trerè D (2000) AgNOR staining and quantification. Micron 31:127-131

Trerè D, Migaldi M, Trentin GP (1995) Higher reproducibility of morphometric analysis over the counting method for interphase AgNOR quantification. Anal Cell Pathol 8:57-65[Medline]

Trerè D, Pession A, Derenzini M (1989) The silver stained proteins of interphasic nucleolar organizer regions as a parameter of cell duplication rate. Exp Cell Res 184:131-137[Medline]

Trerè D, Pession A, Montanaro L, Chieco P, Derenzini M (1997) AgNOR protein expression and tumor growth rate of human carcinoma xenografts growing sub-cutaneously in nude mice. Eur J Histochem 41(suppl 2):153-154

van Diest PJ, Brugal G, Baak JP (1998) Proliferation markers in tumours: interpretation and clinical value. J Clin Pathol 51:716-724[Medline]

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jacquet, B. Articles by Brugal, G. Search for Related Content PubMed PubMed Citation Articles by Jacquet, B. Articles by Brugal, G. Social Bookmarking                      What's this?

Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact