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Nucleolar Size and Activity Are Related to pRb and p53 Status in Human Breast Cancer

Davide Treré, Claudio Ceccarelli, Lorenzo Montanaro, Elena Tosti and Massimo Derenzini

Department of Experimental Pathology, Unit of Clinical Pathology (DT,LM,ET,MD), and Department of Radiological and Histocytopathological Sciences (CC), University of Bologna, Bologna, Italy

Correspondence to: Davide Treré, Alma Mater Studiorum–Università di Bologna, Dipartimento di Patologia Sperimentale, Via San Giacomo 14, 40126 Bologna, Italy. E-mail: davide.trere{at}unibo.it

	Summary

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Cell proliferation is tightly coordinated with cell growth. The oncosuppressor proteins pRb and p53 may exert a key role in coupling growth and proliferation by controlling both ribosome biogenesis and cell cycle progression. In the present study we evaluated the relationship between the pRb and p53 status and rRNA transcriptional activity in histological sections of 343 human primary breast carcinomas. Ribosomal biogenesis was quantified by morphometric analysis of silver-stained interphase nucleolar organizer regions (AgNORs). pRb and p53 status was assessed by immunohistochemistry. Twenty-four tumors were considered to be pRb deleted, 260 tumors showed a phosphorylated-pRb labeling index (LI) up to 25%, and 55 tumors an LI >25%. Tumors with deleted pRb or phosphorylated-pRb-LI 25% were characterized by significantly greater mean AgNOR area values than those with unaltered pRb (p<0.001). In the 71 tumors with mutated p53 the NOR area mean value was greater than in the 272 tumors with normal p53 (p<0.001). Our results demonstrate, for the first time in vivo, that pRb and p53 status is related to the ribosome biogenesis rate and suggest that in tumors with altered pRb and p53 function the up-regulation of rRNA synthesis may always assure an adequate growth to cancer cells with uncontrolled cell cycle progression. (J Histochem Cytochem 52:1601–1607, 2004)

Key Words: pRb • p53 • AgNORs • ribosome biogenesis • breast cancer

	Introduction

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IN CONTINUOUSLY DIVIDING NORMAL CELLS a progressive increase of the cell constituents occurs during the cell cycle phases to avoid a progressive reduction of the daughter cell size. Cell growth and proliferation are therefore two tightly coordinated biologic phenomena that ensure normal cell generations (Thomas 2000). In proliferating cells the increased demand for protein synthesis necessary for cell growth is accomplished by changes in the rate of ribosome biogenesis. Ribosome biogenesis is in fact the major metabolic effort of a proliferating cell (Schmidt 1999). The rate of ribosome biogenesis is linked to and integrated with the proliferative response of the cell and its progression throughout the cycle.

Cell cycle regulator systems have been shown to control rRNA transcriptional activity. Transcription of ribosomal genes depends, at the exit of the mitosis, on reduced mitotic cyclin/Cdk complexes activity (Sirri et al. 2000) and during G1 phase progression on phosphorylation of Upstream Binding Factor (UBF) by G1-specific cyclin/Cdk complexes (Voit et al. 1999). Moreover, inhibitors of cyclin-dependent kinases disrupt nucleolar organization and hinder pre-rRNA processing (Sirri et al. 2002). Mouse liver cells with induced conditional deletion of 40S ribosomal protein failed to proliferate after partial hepatectomy without changes in the ability to synthesize proteins (Volarevic et al. 2000). Normal ribosome biogenesis during G1-phase is necessary for the expression of cyclin E with the consequent formation of the cyclinE/Cdk2 complexes, phosphorylation of the retinoblastoma protein (pRb), and the irreversible transit of the cell throughout the G1-phase restriction point (Volarevic et al. 2000). Moreover, perturbation of normal rRNA processing and ribosome assembly leads to cell cycle arrest in a p53 pathway-dependent manner (Pestov et al. 2001). pRb and p53 appear, therefore, to play an important role both in the control of cell cycle progression and in assuring a cell growth sufficient for the generation of two normal daughter cells. Changes of both the pRb and p53 pathways occur in a variety of human cancers that are responsible for the loss of the normal control mechanisms of cell cycle progression. In this case, the functional relationship between cell growth and cell proliferation might also be expected to be deregulated, with the possibility for cancer cells to divide without having reached an adequate size. However, these altered controls do not hinder intense cell growth and proliferation. On the contrary, cancers with pRb deletion and/or functionally inactive p53 are generally more aggressive than those with normal functioning pRb and p53 pathways. This fact can be explained by the effects of pRb and p53 on ribosome biogenesis. These oncosuppressor proteins repress transcription of the rRNA genes by affecting the activity of UBF (Voit et al. 1997; Budde and Grummt 1999; Hannan et al. 2000a,b; Zhai and Comai 2000). Therefore, cancer cells characterized by deletion or functional changes of pRb or by p53 mutation should also be characterized by a more intense ribosomal biogenesis, thus being prevented from dividing without sufficient growth. To ascertain whether in cancer cells pRb and p53 changes are associated with high levels of ribosome biogenesis, in the present study we have evaluated the relationship between the pRb and p53 status and the quantity of the interphase nucleolar organizer regions (NORs) in histological sections of 343 human breast carcinomas. The NORs are chromosome segments that contain ribosomal genes (Gall and Pardue 1969). During interphase, NORs are located within precise nucleolar structures: the fibrillar centers and the closely associated dense fibrillar component (Bourgeois et al. 1979; Hernandez-Verdun et al. 1980; Hernandez-Verdun 1986). All the components necessary for ribosome gene transcription are located within the interphase NORs where in fact rRNA synthesis does occur (Derenzini et al. 1990). Interphase NORs can be visualized at light microscope level, in routinely processed cytohistological samples, by silver staining a set of proteins selectively associated with them (Hernandez-Verdun et al. 1980). The silver-stained NORs are called AgNORs. The quantity of AgNORs is closely related both to nucleolar size and RNA polymerase I transcriptional activity (Derenzini et al. 1992; Derenzini et al. 1998; Derenzini et al. 2000) and can therefore be used as a parameter for the evaluation of ribosome biogenesis rate in situ. Visualization of the NORs was achieved using the silver staining method originally described by Ploton et al. (1986) and then modified for formalin-fixed samples by Öfner et al. (1994). pRb status was investigated by immunocytochemistry using monoclonal antibodies vs both the phosphorylated and non-phosphorylated form of pRb. Changes of p53 were evaluated by the detection of protein accumulation using the relative monoclonal antibody. In fact, mutate p53 gene products show a prolonged half-life leading to a nuclear accumulation that can be easily detected by immunohistochemistry. Quantitative analysis of all parameters was performed by computer-assisted image cytometry.

	Materials and Methods

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Patients
We studied tumor specimens from 343 consecutive cases of primary invasive breast carcinomas surgically treated at the I Surgical Department and diagnosed at the II Service of Pathology of the S. Orsola-Malpighi University Hospital between 1991 and 1994. Tumors were histologically classified according to World Health Organization criteria and staged according to the UICC–TNM system. Histologically, 306 cases (89.2%) were ductal NOS (not otherwise specified) carcinomas; 29 cases (8.5%) were classified as infiltrating lobular carcinomas, 7 cases (2%) as mucoid carcinomas, and 1 case (0.3%) as a medullary carcinoma. Axillary lymph node metastases were reported as absent (N0) or present (N+). Due to patient age, axillary dissection was not performed in 9 patients: 173 patients (51.8%) were N0 while 161 patients (48.2%) were N+.

Immunohistochemical Assessment
From each case, one block of formalin-fixed, paraffin-embedded tissue was selected, including a representative tumor area. Four µm-thin serial sections were cut, collected on 3-ethoxy-aminoethyl-silane-treated slides and allowed to dry overnight at 37C.

pRb immunostaining was assessed using two different monoclonal antibodies (MAbs): the clone G3-245 (BioGenex Laboratories, San Ramon, CA) which specifically recognizes the phosphorylated pRb form, and the clone 1F8/Rb1 (Neomarkers, Lab Vision, Newmarket Suffolk, UK) which reacts with the hyper-phosphorylated as well as the un- or under-phosphorylated forms of the Rb protein. Before immunostaining, sections were microwaved in EDTA buffer solution (pH 8.0) for 10 min at 750 W. After cooling to room temperature, slides were incubated with primary MAbs overnight at the following dilution: 1:160 for clone G3-245 and 1:30 for clone 1F8/Rb1. The immunostaining reaction was then developed according to the SABC (Stretavidin-Biotin-Peroxidase Complex) method and highlighted using a peroxidase/DAB enzymatic reaction (Santini et al. 1993).

p53 immunostaining was assessed using an anti-p53 MAb (clone BP53-12.1 from BioGenex Laboratories). Before immunostaining, sections were microwaved in 10 mM citrate buffer solution (pH 6.0) for 17.5 min. at 750 W. After cooling to room temperature, slides were incubated with primary MAbs overnight at a dilution of 1:1800. The immunostaining reaction was developed and highlighted as previously described.

NOR Silver Staining
NOR silver staining was performed according to the guidelines of the "International Committee on AgNOR Quantitation" (Treré 2000). In short, slides were moved from water to heat-resistant plastic Coplin jars, fully immersed in 10 mM sodium citrate buffer (pH 6.0) and autoclaved at 120C for 20 min. After cooling to room temperature in the sodium citrate buffer, slides were stained with silver for 13 min at 37C in the dark using a solution of one volume 2% gelatin in 1% aqueous formic acid and two volumes of 50% silver nitrate. Sections were finally dehydrated and mounted in Canada balsam without any counterstaining.

Image Cytometry
Quantitative evaluation of all parameters was carried out by image cytometry in serial sections stained for pRb and p53 and NORs, on corresponding tumor areas of the specimens. p53 and pRb immunostaining was semi-quantitatively assessed using the Cytometrica program (C and V, Bologna, Italy) as previously detailed (Faccioli et al. 1996) and expressed as the percentage of labeled nuclei over total neoplastic nuclear area in the section (labeling index: LI). For each case, at least 2000 cells were evaluated. Morphometric analysis of silver-stained NORs was carried out using the Image-Pro Plus program (Media Cybernetics, Silver Spring, MD). The main stages of image processing were as follows: a field was selected by the operator at the microscope under a x40 objective lens. The selected image was then captured and stored in the digital memory and displayed on the color monitor. Here the operator interactively defined the gray threshold that permitted automatic quantification of only the black dots corresponding to the silver-stained nucleoli. The morphometric analysis was then performed on a cell-to-cell basis by converging the window over the corresponding nucleus. For each case, the AgNOR area of at least 200 nuclei was measured and the mean AgNOR area (±SD) calculated.

Statistical Analysis
Correlations between parameters considered as continuous variables were analyzed using the Spearman rank correlation test. Differences between categorical variables were analyzed using the ANOVA test. Values for p<0.05 were regarded as statistically significant.

	Results

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Evaluation of Ribosome Biogenesis Rate by AgNOR Quantification
In the present study, quantitative evaluation of interphase AgNORs was carried out by morphometric analysis, by measuring the area occupied by the silver-stained structures (see above). This method has been demonstrated to be more reliable than the direct count of the AgNOR number, due to the difficulty of separating single AgNORs from each other at light microscopic level (Treré 2000). In our series, the mean AgNOR areas ranged from 1.63 to 11.63 µm², with a mean (±SD) value of 4.12 (±1.63) µm² and a median value of 3.72 µm².

Relationship between the Status of p53 and pRb and the AgNOR Quantity
In our series the percentage of p53 immunostained cells ranged from 0 to 99.7, with a mean ± SD value of 15.8 ± 29.4. We considered samples with at least 10% nuclear activity to be p53 positive (+). This cutoff value was selected according to Esrig et al. (1993), who demonstrated a strong correlation of mutation in the p53 gene with the accumulation of p53 protein in 10% or more of the tumor cell nuclei. Among the 343 cases evaluated, 272 (79.3%) showed a p53-LI lower than 10% while the remaining 71 (20.7%) presented nuclear accumulation of p53 (LI 10%). The AgNOR area of the group of tumors showing p53 accumulation was greater (mean ± SD: 5.34 ± 1.72 µm²) than that of tumors with p53-LI <10% (mean ± SD: 3.81 ± 1.46 µm²), with a highly significant difference between the two groups (F = 58.58; p<0.001). The positive correlation between AgNOR and p53 values was also maintained when the two parameters were considered as continuous variables (r = 0.33, p<0.001). Two breast carcinomas characterized by a different p53 status are reported in Figure 1. In Figure 1a, a case is shown with an evident nuclear accumulation of p53: most cancer cells are immunostained while stromal cells and lymphocytes (regarded as negative internal controls) appear negative. In Figure 1b, another breast carcinoma is reported, showing only a few immunostained cancer cells. Figures 1c and 1d show the same cases reported in Figures 1a and 1b, after silver staining. The AgNOR area is greater in cancer cells of the case with p53 accumulation (Figure 1c) than in cancer cells with a low p53-LI (Figure 1d).

View larger version (126K): [in this window] [in a new window]   Figure 1 p53 immunostaining and AgNOR staining of two breast carcinomas. p53 accumulation is evident in (a) (p53-LI = 97.1%), while only a few cancer cells are positive in (b) (p53-LI = 2.6%). Note the greater AgNOR quantity in the cancer cells shown in (c) (mean AgNOR area = 7.27 µm2) as compared with that of cancer cells shown in (d) (mean AgNOR area = 3.71 µm2).

For statistical analysis, the pRb variable was separated into three groups, indicative of three different pRb states: the first group (pRb-0) included the 24 cases with a presumed deletion of the pRb gene, the second group (pRb-1) included 260 cases with low pRb immunostaining (pRb-LI <25%), and the third group (pRb-2) included 55 cases with a high pRb immunostaining (pRb-LIs >25%). The 25% cutoff value was chosen considering that pRb hyperphosphorylation characterizes mainly late G1-, S- and G2-phases, whose duration in human cancers is not longer than 1/4 of the cell cycle length (Rew and Wilson 2000). Therefore, the presence of a pRb-LIa >25% is strongly indicative of an alteration of the pRb phosphorylation control. Moreover, the value of 25% was found to represent the best cutoff for discriminating between two groups of patients at different prognoses in disease-free survival analysis (Derenzini et al. 2004). The mean AgNOR area values were 5.74 ± 1.34 µm² in the pRb-0 group, 3.69 ± 1.35 µm² in the pRb-1 group and 5.44 ± 1.88 µm² in the pRb-2 group. ANOVA analysis highlighted highly significant differences in the mean AgNOR area values between pRb-0 and pRb-1 groups (F = 50.82; p<0.0001) as well as between pRb-1 and pRb-2 groups (F = 63.83; p<0.0001), while no significant difference was found between the pRb-0 and pRb-2 groups (F = 0.50; p = 0.485). When both pRb-LI and AgNOR areas were analyzed as continuous variables, excluding the 24 pRb-0 cases, a significant correlation was found (r = 0.420; p<0.0001).

Figures 2a and 2b report two breast carcinomas immunostained with the monoclonal antibody which specifically recognizes the phosphorylated pRb form (clone G3-245): only a few cancer cells are positive in Figure 2a, while a greater number of nuclei are positive in Figure 2b. In both cases, as expected, non-cancerous cells are negative. Figure 2c reports a breast carcinoma with a presumable pRb deletion. In this case, pRb immunostaining was carried out using the monoclonal antibody that recognizes the total pRb form (clone 1F8/Rb1): cancer cells are absolutely negative for total pRb, while stromal cells (which we have considered as positive internal controls) are clearly immunostained. In Figures 2d, 2e and 2f, the same cases reported in Figures 2a, 2b and 2c are shown after selective silver staining for NOR visualization. Compare the small AgNOR area of cancer cells in the case with a low phosphorylated pRb-LI (Figure 2d) with the greater AgNOR area in the cases with a high phosphorylated pRb expression (Figure 2e) and pRb deletion (Figure 2f).

View larger version (149K): [in this window] [in a new window]   Figure 2 pRb immunostaining and AgNOR staining of three breast carcinomas. pRb immunostaining was obtained by two different MAbs. Clone G3-245, which specifically recognizes the phosphorylated pRb form, was used in (a) and (b), while clone 1F8/Rb1, which reacts with total pRb, was applied in (c). Note the different phosphorylated pRb expression between (a) and (b) (pRb-LIs: 2.7% and 34.5%, respectively), and the negative pRb immunostaining of cancer cells in (c), indicating a presumable deletion of the pRb gene. (d), (e) and (f) show the AgNOR staining of the three cases reported in (a), (b) and (c), respectively. The mean AgNOR area was 3.75 µm2 in (d), 5.88 µm2 in (e), and 6.34 µm2 in (f).

	Discussion

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The retinoblastoma protein is a key negative regulator at the G1-phase restriction point that controls the passage of a cycling cell to an irreversible commitment to division. pRb exerts its function by binding the E2F family of transcription factors, thus preventing their activity. These factors stimulate the expression of a series of genes involved in the restriction point overriding and the progression throughout the S-phase (Harbour and Dean 2000). The suppressive role of pRb at the restriction point is inactivated by its phosphorylation by cyclin E-cdk2 complexes (Ezhevsky et al. 2001) that disrupt its association with the E2F factors. There is evidence that pRb, in addition to the control role on cell cycle progression, also regulates the cell growth by modulating the synthesis of rRNA (Cavanaugh et al. 1995). It was shown that pRb acts as a transcriptional repressor by interfering with the assembly of transcription initiation complexes. The inhibitory effect on RNA polymerase I transcription was the consequence of the binding of hypophosphorylated pRb to UBF (Voit et al. 1997; Hannan et al. 2000a,b). Along the cell cycle the sequential activation of the cyclin-dependent kinases progressively increases the degree of pRb phosphorylation from early G1, during which it is hypophosphorylated, to G2-phase when pRb is hyperphosphorylated (Donjerkovic and Scott 2000). Accordingly, the rRNA transcription rate progressively increases from G1- to G2-phase. pRb appears therefore to control not only the cell cycle progression but also that cycling cells grow enough to divide without ever becoming smaller. When changes of pRb status alter its function, the cross-talk between proliferation and growth is not interrupted. Our results have in fact shown that breast cancers—in which the control activity of pRb on restriction point is lost or reduced by either deletion or hyperphosphorylation of the oncosuppressor protein—are characterized by a very high ribosome biogenesis rate, very likely due to the loss of the pRb inhibitory effect on the RNA polymerase I activity. This intense ribosome biogenesis may allow the cell to reach, at the end of the cycle, an adequate size to give rise to two normal daughter cells independently of the length of the cell cycle that may be reduced as a consequence of the absence of a normally functioning restriction point control.

The interaction between growth and proliferation is also controlled by p53, the other canonical oncosuppressor protein. p53 is a transcription factor induced by DNA damage or inappropriate mitogenic signaling which induces cell cycle arrest at G1-phase or apoptosis (Levine 1997). Recently, it has been demonstrated that changes of normal ribosome biogenesis can also activate p53 to trigger a cell cycle-inhibitory response. Strezoska et al. (2000) found that a mutant form of Bop1, a nucleolar protein involved in rRNA processing and ribosome assembly, completely blocks formation of the mature 28S and 5.8S rRNA, which results in a deficiency of the 60S ribosome sub-units and arrest of the cell cycle. Asynchronously proliferating cells expressing the mutant form of Bop1 are arrested in the G1-phase for their inability to progress through the restriction point. The cell cycle arrest is dependent of the normally functioning p53 (Pestov et al. 2001). p53 appears therefore to monitor ribosome biogenesis to avoid a cell cycle progression without optimal growth conditions. Mutations of p53 with the loss of its function are the most common alterations in human cancer. Once again a neoplastic cell lacking functional p53 may pass throughout the restriction point regardless of the metabolic conditions of the cell. However, as far as ribosome production is concerned, cells lacking functional p53 are preserved from progressing the cell cycle without a sufficient growth support by a very high ribosome biogenesis rate. Our results have in fact demonstrated that breast cancers with mutated p53 are characterized by nucleoli intensely active in rRNA synthesis. This finding is consistent with the inhibitory effect of p53 on RNA polymerase I activity: wild type but not mutant p53 hinders RNA polymerase I transcription by binding to the selectivity factor SL1 (Zhai and Comai 2000).

The observation that breast cancers with deleted pRb or high phosphorylated-pRb labeling index or mutated p53 are characterized by a greater AgNOR quantity than those with normal pRb and p53 status allows some considerations to be made on the relationship between nucleolus and cancer.

For a long time it has been known that hypertrophied and irregularly shaped nucleoli frequently characterize malignant cells and that these features have also been exploited for tumor diagnosis (Busch and Smetana 1970). However, since all these nucleolar changes were also present in rapidly growing cells of embryonic, regenerating and glandular tissues (Bernhard and Granbulan 1968), interest in the nucleolus and its functions in relation to the processes involved in cell transformation has been very low for many years. Recently, after a series of studies indicating that the mechanisms that control cell proliferation also control ribosome biogenesis, the question has been clearly posed of how and to what extent tumor suppressors and oncogenes modulate ribosome function and of whether a change in this function is the cause or the consequence of cancer progression (Ruggero and Pandolfi 2003). Our results indicate that, at least for breast cancer, nucleolar hypertrophy and high ribosome biogenesis rate are the consequence of the loss of the inhibitory effects of pRb and p53 on RNA polymerase I activity. On the other hand, this up-regulation of rRNA synthesis may be considered a mechanism by which cancer cells with deregulated cell cycle progression can always reach, at the end of each cycle, a size large enough to divide without ever becoming smaller until they can no longer proliferate. Therefore, in light of this fact, a high ribosome biogenesis rate would be necessary for progression of cancers with altered pRb and p53 functions.

	Acknowledgments

 
This work was supported by grants from Pallotti's Legacy for Cancer Research, MIUR (Ministero dell'Istruzione, dell'Università e della Ricerca; finanziamenti per la Ricerca Fondamentale Orientata) and University of Bologna.

	Footnotes

 
Received for publication June 23, 2004; accepted August 19, 2004

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