Intranuclear Nucleolin Distribution during Cell Cycle Progression in Human Invasive Ductal Breast Carcinomas in Relation to Estrogen Receptor Status

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1 Intranuclear Nucleolin Distribution during Cell Cycle Progression in Human Invasive Ductal Breast Carcinomas in Relation to Estrogen Receptor Status MAREK MASIUK, ELZBIETA URASINSKA and WENANCJUSZ DOMAGALA Department of Pathology, Pomeranian Medical University, Szczecin, Poland Abstract. Background: Nucleolin (NU) can be found in the nucleus in aggregates and in a diffused form. The expression and distribution of these two intranuclear pools of nucleolin was compared in estrogen receptor (ER)-positive and -negative invasive ductal breast carcinomas in relation to cell cycle phases. Materials and Methods: Using a laser scanning cytometer NU was measured within the whole nucleus, within nucleolin aggregates (NUA) and within the NUA-free karyoplasm. The G 1 -, S- and G 2 M-phase cell subpopulations were distinguished on DNA histograms. Results: The nuclei of ER-positive and ER-negative breast carcinomas in the G 2 M- phase differed in the expression of NU in the NUA and in the NUA-free karyoplasm, whereas in the S-phase they only differed in the NUA-free karyoplasm expression. In ER-positive carcinomas NU levels rose from G 1 to G 2 M in both the NUA and the NUA-free karyoplasm, whereas in ER-negative carcinomas the NU levels increased only between G 1 /S in the NUA and between S/G 2 M in the NUA-free karyoplasm. Conclusion: There are significant differences in nucleolin expression and localization during cell cycle progression depending on ER status. The nucleolus is formed by the nucleolar organizer and contains genetic information for ribosomal RNA (1). It disappears during metaphase and reappears in the early G 1 - phase (2). The main function of the nucleolus is rrna processing from transcription to assembly of preribosomal particles. Nucleolin (NU) is one of most abundant nucleolar proteins comprising about 10% of its proteins (3). It possesses three domains: the N-terminal domain, rich in acidic amino acids, which is involved in silver staining of Correspondence to: Marek Masiuk, MD, Ph.D., Department of Pathology, Pomeranian Medical University, ul. Unii Lubelskiej 1, , Szczecin, Poland. Tel/Fax: , mmasiuk@med.pam.szczecin.pl Key Words: Nucleolin, cell cycle, estrogen receptor, nucleolus, ductal breast cancer. nucleolin (AgNOR argyrophilic nucleolar organizer regions) (4), the central, RNA-binding domain (5) and the glycine-rich C-terminal domain (6). Nucleolin is mainly located in fibrillary centers and dense fibrillary components of the nucleoli (7) and in nucleolar organizers in the chromosomes (8). Between prometaphase and the middle of telophase, nucleolin has been detected in the cytoplasm (9, 10). In interphase nuclei it has been detected in both subnuclear compartments, the nucleoli and the karyoplasm outside the nucleoli (3, 7, 8), and this distribution pattern has been strongly supported by immunofluorescence studies (9-11). The main function of nucleolin is participation in ribosome biosynthesis (7, 12, 13). Nucleolin preferentially binds to glycine-rich rdna (12), influencing the chromatin structure by interacting with histone H1 (14). It also takes part in early pre-rrna processing as well as in recruitment of other factors forming a primary processing complex (15, 16), and it affects further stages of maturation of pre-rrna by interactions with proteins such as B23 or fibrillarin (3). The estrogen receptor (ER) is a proteinaceous transcription factor binding DNA. Ligand binding by the estrogen receptor leads to conformational changes of the receptor, its dimerization, interaction with DNA and coactivation of other transcription factors (17, 18). ER expression has been detected in 69-85% female breast carcinomas (19). ERpositive carcinomas differ from ER-negative ones in histopathology and prognosis. Among common breast cancer types, high-grade invasive ductal and medullary carcinomas are mostly ER-negative (20, 21) while low-grade ductal and lobular carcinomas are generally ER-positive (22, 23). ER expression is a predictive factor (24, 25) with approximately 60-70% ER-positive breast cancer patients but less than 10% of those with ER-negative carcinomas responding well to hormonal therapy (25). In both normal breast tissue and cancer cells estrogens play a role in the regulation of entrance to S-phase by increasing c-myc and cyclin D1 expression followed by cyclin E-Cdk2 activation and prb phosphorylation (26). A posititive correlation between the ER and cyclin D1 has been found in normal breast tissue and in breast carcinomas (27, 28). AgNOR proteins have /2007 $

2 been described in human breast cancer in relation to ER status (29-31) or the cell cycle (32-34). However, there are no published reports of nucleolin expression in the different intranuclear pools in ER-positive and -negative human breast carcinomas in relation to the cell cycle. In the present study the expression and distribution of two different intranuclear pools of nucleolin that is within the nucleolin aggregates (nucleolar) and in the remaining karyoplasm (nucleoplasmic) was compared in ER-positive and -negative invasive ductal breast carcinomas in relation to cell cycle phases. Materials and Methods The studies were performed on tumor cells from 87 women with primary, invasive, ductal breast carcinomas who had undergone surgery between September, 2002, and December, 2003, in the District Oncologic Hospital in Szczecin, Poland. No chemo- or radiotherapy was applied prior to surgery. In each case the histological type, histological grade according to Bloom and Richardson (35), lymph node status and estrogen receptor status were assessed in formalin-fixed, paraffin-embedded tissue. The mean age of patients was 57.5±14.1 years and the mean tumor diameter was 20.9±8.2 mm. Axillary lymph node metastases were found in 45 (52%) patients. Six tumors were of grade I (7%), 38 tumors were of grade II (44%) and 43 were of grade III (49%). There were 67 (77%) ER-positive and 20 (23%) ER-negative tumors. The measurements were performed in cytospins of breast cancer cells obtained by scratching a cut-surface of a tumor with a blade in PBS (phosphate-buffered saline). The suspension of cells was cytocentrifuged (1000 rpm, 6 minutes, Shandon, Shandon Scientific Ltd., England). The cytospin slides were fixed in 1% solution of buffered formalin (15 minutes on ice) followed by 70% ethanol ( 20ÆC for 24 hours). The slides were air-dried at room temperature and stored at 20ÆC. The NU expression was detected by immunofluorescence with monoclonal mouse anti-human nucleolin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) diluted 1:20 in PBS followed by FITC (fluorescein isothiocyanite)-conjugated F(ab ) 2 fragments of goat anti-mouse antibody (DAKO Corp., Carpinteria, CA, USA) diluted 1:20 in PBS. Nuclear DNA was counterstained with a solution of 5 Ìg/ml propidium iodide (PI) (Molecular Probes, Eugene, OR, USA) in the presence of 100 Ìg/ml RNaze A (Sigma-Aldrich, Poznan, Poland) in PBS. All the incubations were performed for 30 minutes, at room temperature, in the dark. As a negative control an isotype antibody was used instead of the primary antibody (DAKO Corp., USA). Fluorescence parameters were measured in at least 10,000 tumor cells by a laser scanning cytometer (Compucyte Corp., Cambridge, MA, USA) with an argon laser. The green FITC NU-associated fluorescence (maximum of emission 520 nm) and red PI DNAassociated fluorescence (maximum of emission 610 nm) parameters were analyzed. FISH (fluorescence in situ hybridization) application of WinCyte 3.4 software (CompuCyte Corp., USA) was used for estimation of the NU aggregates (NUA). The spots of green fluorescence of intensity above 900 arbitrary units and area of 1 Ìm 2 were considered NUA. The expression of NU was measured in three nuclear compartments; the whole nucleus, the NUA and the NUA-free karyoplasm. The nuclear area (Ìm 2 ), the area of NUA Figure 1. Schematic approach to analysis of NU expression in relation to cell cycle phases in ductal breast carcinoma by a laser scanning cytometer. (area of all NUA within the nucleus, Ìm 2 ) and the area of NUAfree karyoplasm (Ìm 2 ) were analyzed based on the projection of PI red fluorescence and NU-associated green fluorescence on the surface of the slide and the number of NUA was counted. The following parameters of fluorescence were analyzed: i) the mean total nuclear NU fluorescence (nuclear NU); ii) the mean NU fluorescence in the NUA (NU in NUA); iii) the mean NU fluorescence in the NUA-free karyoplasm; iv) the ratio of the mean total nuclear NU fluorescence to the mean nuclear area (Ìm 2 ); v) the ratio of the mean NU fluorescence in the NUA to their area (Ìm 2 ); and vi) the ratio of the mean NU fluorescence in the NUAfree karyoplasm to its area (Ìm 2 ). The approach to analysis of NU expression in relation to the cell cycle phases is presented in Figure 1. A single-cell population was distinguished based on the value of the maximal pixel of red PI fluorescence and the nuclear area. The G 1 -phase, S-phase and G 2 M-phase cell subpopulations were distinguished and the mean NU-associated fluorescence in the nuclei and in the NUA was analyzed. The mean NU-associated fluorescence in the NUA-free nucleoplasm was calculated by subtraction of the mean NUassociated fluorescence in NUA from the mean NU-associated fluorescence in the nucleus. Formalin-fixed and paraffin-embedded tumor sections were dewaxed in xylene and rehydrated in ethanol, and the ER expression was detected immunohistochemically. The slides were incubated with biotinylated monoclonal anti-human ER antibody (DAKO Corp.) diluted 1:50 and visualized by streptavidin-biotin reaction using DAKO LSAB (labeled streptavidin biotin) + System, HRP (horseradish peroxidase) (DAKO Corp.). Then nuclei were stained with hematoxylin. At least 1,000 nuclei were counted using a 40x objective and an automated image analysis system, Leica Q600 QWin (Leica Cambridge Ltd., UK). Tumors with at least 10% of positively stained nuclei were considered ER-positive. 3958

3 Masiuk et al: Nucleolin, Cell Cycle and Ductal Breast Carcinoma Table I. Nucleolin expression (fluorescence intensity) in nuclei of 87 invasive ductal breast carcinomas in relation to ER status (mean±sd). Parameter All carcinomas (n=87) ER-negative (n=20) ER-positive (n=67) p* NU in nucleus ± ± ± NU in NUA ± ± ± NU in the NUA-free karyoplasm ± ± ± Ratio of nuclear NU to its area 4747± ± ± Ratio of NU in NUA to its area 30491± ± ± Ratio of NU in the NUA-free karyoplasm to its area 2221± ± ± *P-value shows differences between ER-negative and ER-positive carcinomas. NU: nucleolin. NUA: nucleolin aggregates; Table II. Nucleolin expression in nuclei of invasive ductal breast cancer cells in relation to ER-status in cell cycle phases (mean±sd). Cell cycle phases All carcinomas (n=87) ER-negative (n=20) ER-positive (n=67) p* Nuclear compartment Total nucleus G ± ± ± S ± ± ± G 2 M ± ± ± NUA G ± ± ± S ± ± ± G 2 M ± ± ± NUA-free karyoplasm G ± ± ± S ± ± ± G 2 M ± ± ± *P-value shows differences between ER-negative and ER-positive carcinomas. NUA: nucleolin aggregates. The differences between values were calculated using the Mann- Whitney U-test. The analysis of the NU expression in the cell cycle phases in relation to ER-status was conducted in a two-step pattern. First, the differences of NU expression in a certain nuclear compartment in the G 1 -, S- and G 2 M-phases were calculated using the Friedman ANOVA test, then the differences of NU expression between phases in a certain nuclear compartment were calculated using the Wilcoxon s test. All analyses were performed using Statistica 5.0 software. The p-values less than 0.05 were considered significant. Results The mean nuclear area of the ER-negative carcinomas was higher (78.5±14.2 Ìm 2 ) than that of ER-positive ones (71.3±18.0 Ìm 2 ) (p=0.04). Despite wide variations in total nucleolin expression among breast cancer cells (as attested by high SD) the mean nucleolin expression in the nuclei of ER-positive breast cancer cells was higher than that in ERnegative carcinomas (p=0.05) (Table I). The number of NUA, the area of the NUA and the area of the NUA-free karyoplasm did not differ significantly between ER-positive and -negative carcinomas (data not shown). During progression of the cell cycle (from G 1 to G 2 M) a 1.8-fold increase of total nucleolin expression within the nuclei of all analyzed carcinomas was observed (Table II), which was due mainly to the increase of expression of nucleolin in the karyoplasm because the expression of nucleolin in the aggregates remained stable between S- phase and G 2 M-phase and increased significantly only between the G 1 - and S-phases (Figure 2). Total nuclear nucleolin expression increased 1.88-fold in ER-positive and 1.46-fold in ER-negative carcinomas. The ER-positive ductal carcinomas showed a significantly higher ratio of nucleolin to nuclear area (p=0.007) (Table I), higher total nucleolin expression in the nucleus and in the NUA-free karyoplasm in S-phase cells (p=0.004 and p=0.002 respectively), in G 2 M-phase cells (p=0.001 and p=0.004 respectively), and in the NUA in G 2 M-phase cells (p=0.04) than the ER-negative carcinomas (Table II). The ER-positive carcinomas showed a significant increase of total NU expression in the whole nucleus and in both nuclear compartments between all cell cycle phases (Figure 3A) whereas the ER-negative carcinomas showed an increase of total nuclear NU expression only between the G 1 and S (p<0.001), and G 1 and G 2 M phases (p<0.001), an increase of NU expression in NUA only between G 1 and S phases (p<0.001), and an increase of NU expression in the NUA-free karyoplasm only between G 1 and G 2 M (p<0.01) and S and G2M phases (p<0.001) (Figure 3B). 3959

4 Figure 2. Nucleolin expression in nuclei of all analyzed ductal breast cancer cells during the cell cycle progression. Discussion Nucleoli have been studied by assessment of rdna (36), by AgNOR (28, 37, 38) or by their constituent proteins, such as nucleolin (9, 11), in various cells using image analysis, confocal microscopy or laser scanning cytometry. The morphometric studies of AgNOR proteins in breast lesions have focused on analyses of the size of the silver-stained dots related mainly to nucleolin and B23 protein, neglecting the possible expression of these proteins in the remaining karyoplasm (28, 30, 33, 37, 39). Moreover studies implementing blotting techniques have been limited to general, nucleolar detection of nucleolin without analysis of subnuclear fractions (34, 40). Variability of staining depending on the time and conditions of incubation have led to low comparability between different studies (41, 42). The aggregated nucleolin participates in rdna processing as it is colocalized with rdna in the nucleoli and subsequently associates with pre-rrna (12, 13). In the present study nucleolin expression and nuclear nucleolin concentration was higher in ER-positive breast carcinomas than in ER-negative ones. Nucleolin is considered to be an AgNOR protein (4). Raymond and Leong found a rather weak, inverse correlation between the number of AgNORs and the percentage of ER-positive cells (31) however Ruschoff et al. found a lower number and mean area of AgNORs in ER-negative breast carcinomas (29) and Gunther et al. reported a higher number of AgNORs and larger total AgNOR area in ER-positive cells (30). The latter results partially correspond to our findings of higher nucleolin expression in ER-positive carcinomas although we found no significant differences in nucleolin expression in aggregates or in the number or area of nucleolin aggregates between the ER-positive and -negative Figure 3. A) Nucleolin expression in nuclei of ER-positive ductal breast cancer cells in cell cycle phases. B) Nucleolin expression in nuclei of ERnegative ductal breast cancer cells in cell cycle phases. carcinomas. Thus the observed difference might have been due to variation in nucleolin concentration in the NUA-free compartment of the nuclei and not to differences in the number of NUAs. These results correspond to those of Sacks et al. (43) and Nakayama et al. who found no correlation between the number of AgNORs and the percentage of ER-positive cells in invasive breast cancer (39). In AgNOR analysis nucleolin is represented only by the area of the AgNORs (40) so the nucleolin present in the AgNORs-free karyoplasm cannot be assessed. Although LSC software limits the contouring of aggregates to as low as 1 Ìm 2, smaller particles can be measured as a fluorescence within the NUA-free karyoplasm (9, 11). Assuming that the area of an AgNOR is Ìm 2 our results on NUAs can be compared with published data on AgNORs (29). Several studies have shown that the expression of nucleolin either as a single protein (9, 34, 40) or as a component of AgNOR (32, 33) is related to the cell cycle. We found a 1.8-fold increase of total nucleolin expression 3960

5 Masiuk et al: Nucleolin, Cell Cycle and Ductal Breast Carcinoma within the whole nucleus between G 1 - and G 2 M-phases. This increase was smaller in the ER-negative carcinomas (1.46-fold) and higher in the ER-positive ones (1.88-fold). Sirri et al. showed a 2-fold increase of nucleolin expression in rat HTC (hepatoma) cells between G 1 - and G 2 -phase, and found that G 0 cells showed very low expression of nucleolin (34). Similar observations have been made by Gorczyca et al. in stimulated human lymphocytes (9). They also reported a slight decrease of nucleolin expression when the cells entered S-phase. Between S-phase and G 2 M we found no increase of nucleolin expression in the NUA but a concomitant increase of nucleolin expression in the NUA-free karyoplasm and in the whole nucleus. This may be due to translocation of nucleolin from the nucleoli to the nucleoplasm at the beginning of the mitotic division. Ochs et al. showed nucleoplasmic distribution of nucleolin as the nucleolus disappeared and subsequent disappearance of nucleolin during metaphase and anaphase with reappearance in telophase (10). The assessment of cell populations in different stages of the cell cycle based on PI fluorescence histograms limits the discrimination of G 2 -phase cells from mitotic cells. Moreover, binding of PI to DNA depends on the level of condensation of the chromatin (44) and contouring of nuclei by LSC software is based on a set threshold value of red PI fluorescence (45). The condensation of chromatin to chromosomes during mitosis influences the ability of PI to intercalate double-stranded DNA and hence the ability of LSC to contour the disintegrating nucleus in prophase. Thus, because the level of PI fluorescence may be too low in some cells they may not be contoured and measured making the precise analysis of expression and distribution of nucleolin in mitotic cells impossible. In our study the population of G 2 M-phase cells, that is the population of cells that is well-recognized in cytometric analyses (and sometimes designated as G 2 - phase) (9, 34, 36) was measured. Interestingly the nuclei of ER-positive and -negative breast carcinomas in G 2 M-phase differed in the expression of nucleolin in the NUA and in the NUA-free karyoplasm, whereas in S-phase differences only occurred in the NUAfree karyoplasm. This may suggest different involvement of nucleolin present in aggregates and in karyoplasm in the S- and G 2 M-phases of the cell cycle depending on ER status. We conclude that ER-positive and ER-negative breast carcinomas differ in expression of nucleolin. There are significant differences in nucleolin expression during cell cycle progression mainly due to its expression in the karyoplasm and not in nucleolin aggregates, depending on the ER status. 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