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ORIGINAL ARTICLE  
Year : 2012  |  Volume : 29  |  Issue : 2  |  Page : 111-115
Can the centrosome be a marker for DNA ploidy in breast cancer?


1 Department of Gynecology, Hopital Tenon, University Pierre et Marie Curie, Paris, France
2 Department of Cytology, Hopital Tenon, University Pierre et Marie Curie, Paris, France

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Date of Web Publication12-Jun-2012
 

   Abstract 

Background: The role of DNA ploidy in genomic instability of cancer cells and prognosis has been described in a number of studies. The role of the centrosome in cell cycle has also been reported.
Aim: In this study, we aimed to investigate the correlation between the centrosome and DNA ploidy in breast cancer in a search for a cytologic predictive and prognostic marker.
Materials and Methods: Cell prints were prepared from cell culture of mesothelial cells, fibroblast cell line MRC5 and breast cancer cell lines MCF7 and T47D. Indirect immunofluorescence was used with anti-γ-tubulin and centrosomes were quantified using a fluorescence microscope. DNA ploidy was scored with the DNA index analyzed by flow cytometry.
Results: The normal mesothelial cells (94% of the cells with one detected centrosome) and MRC5 diploid cells (68% with two centrosomes) were used as quality controls. A correlation between the number of centrosomes and DNA ploidy was found in MCF7 cell lines (64% of the cells with a number of centrosomes ≥ 3). It was not observed in invasive breast cancer samples; however, the frequency of cells with centrosomes ≥ 3 was found to be slightly higher in DNA aneuploid samples than in DNA diploid samples (15% vs 13.3%).
Conclusion: Quantification of centrosome appears to be correlated to DNA ploidy in breast cancer cell lines and slightly associated to DNA aneuploidy in invasive breast cancer. Studies analyzing a larger number of samples as well as morphological abnormalities of the centrosome are needed.

Keywords: Breast cancer; centrosome; DNA ploidy

How to cite this article:
Sakr RA, Fleury J, Prengel C, Bernaudin JF, Uzan S, Rouzier R, Darai E. Can the centrosome be a marker for DNA ploidy in breast cancer?. J Cytol 2012;29:111-5

How to cite this URL:
Sakr RA, Fleury J, Prengel C, Bernaudin JF, Uzan S, Rouzier R, Darai E. Can the centrosome be a marker for DNA ploidy in breast cancer?. J Cytol [serial online] 2012 [cited 2020 Sep 22];29:111-5. Available from: http://www.jcytol.org/text.asp?2012/29/2/111/97150



   Introduction Top


In breast cancer, a significant number of patients will relapse in spite of relevant prognostic factors used for a better targeted therapy. [1],[2],[3] Hence, there was a need to expand the list of identified predictive factors.

Recent studies have shown the importance of DNA ploidy, the reflection of chromosomal instability of cancer cells as predictor of recurrence. [4],[5],[6],[7],[8],[9],[10] Centrosomic abnormalities are also known to be implicated in aneuploidy and genomic instability.

The aim of our study was to explore centrosome abnormalities in breast cancer in a search for a cytological tool analyzing the relationship between DNA ploidy and the number of centrosomes.


   Materials and Methods Top


Control cell lines

As control for normal diploid cells, epithelial cells were extracted from non-carcinomatous ascites in accordance with the ethical standards. First the tubes containing ascites were concentrated by centrifugation. Then the supernatant was poured off and the pellets were washed several times in phosphate buffered saline (PBS). After lysis of red blood cells, mesothelial cells were suspended in PBS. They were cultured in a medium comprising Roswell Park Memorial Institute (RPMI) 1640 (Gibco) and Dulbecco's modified Eagle's medium (DMEM) 1880 (Gibco) supplemented with 10% fetal calf serum (FCS) (Gibco), L 5 mM-glutamine (Sigma), penicillin 50 U/ml (Sigma), streptomycin 50 μg/ml (Sigma) and 20 mM Hepes (Gibco), then incubated in 5% CO 2 at 37°C. Once the cells reached 90% confluence, they were trypsinized at 37°C for 5 to 10 min (trypsin-EDTA 1 X, Gibco), then suspended in an equal volume of culture medium. Cell suspensions were then concentrated by centrifugation (1400 rpm/4 min) at room temperature. Again the supernatant was poured off and the cells were re-suspended in PBS. As control for the diploid cells in cycle, fibroblasts from cell line MRC5 were analyzed at 30%, 60% and 90% confluence. As control for aneuploid cells, breast cancer cell lines MCF7 and T47D were analyzed.

Immunocytochemistry

Breast tissue samples were collected from lumpectomy and mastectomy specimens with the approval of the ethics committee. The presence of invasive carcinoma was confirmed by hematoxylin and eosin (H and E) staining before samples were snap freezed in liquid nitrogen and stored at -80°C. After monolayer cell prints were made, one slide was stained with May-Grünwald-Giemsa (MGG) for cytology diagnosis, and the other two slides were air dried for 24 hours, then stored at -20°C without fixation. Cell suspensions were concentrated to 10 6 cells/ml and pellets were prepared (cytospin 100 μl at 300 rpm for 6 min). For each cell type, one slide was stained with MGG and the remaining slides of each cell type were air dried overnight and stored at -20°C until use.

We used indirect immunofluorescence. The prepared slides (cell prints or pellets) were thawed for five minutes at room temperature. Mesothelial cells were used as control for normal diploid cells, fibroblasts MRC5 diploid cells as control for cells in cycle and tumor cell lines MCF7 and T47D as control for DNA aneuploid tumor cells. The cells of interest were encircled with a waterproof pen (DakoCytomation), before they were fixed in acetone (+4°C for 10 min), and permeabilized in Triton X-100 0.1% for 5 min, at room temperature. Blocking solution (30% normal goat serum in PBS) was applied to the slides for 30 min. Then slides were incubated with mouse monoclonal antibody (Ab) anti-γ-tubuline (1.6 mg/ml, diluted 1:300, Sigma) for one hour at room temperature. After washing in PBS for 10 min, the slides were incubated with the secondary antibody goat anti-mouse FITC-IgG, F(ab') 2 (1.4 mg/ml, diluted 1:50, Immunotech) for 60 min. Counterstaining was performed by incubation with 4×-6-diamidino-2-phenylindole (DAPI) for 1 min, after washing the slides in PBS for 10 min. The slides were then mounted in Vectashield® Mounting Medium (1.5 mg/ml, Vector) and examined under a fluorescence microscope (Leica DM 4000B, Wetzlar, Germany) at magnification ×10, ×60 and ×100 using a blue filter for DAPI (λ = 359 nm, λ = 461 nm) and a green filter for FITC (λ = 490 nm, λ = 525 nm). The images were digitised using a cooled CCD camera (Cool Snaps cf ), and a software for image capture (Genikon, Alphelys, Plaisir, France). In each slide, the number of green spots (centrosomes) per cell was counted in at least 100 cells.

Flow cytometry

Flow cytometry (FCM) was performed in tumor samples that were snap frozen in liquid nitrogen and stored at -80°C. After staining, cell prints with MGG to confirm the presence of tumor cells, samples were mechanically dissociated and the cells were collected in two tubes. In the first tube, human lymphocytes (2.10 5 cells for each 10 6 tumor cells) were added as normal diploid controls and cells were incubated with propidium iodide (DNA prep kit, Beckman Coulter). The analysis of at least 2.10 4 cells was performed using a flow cytometer (Epics Elite® , Beckman Coulter) with argon laser (λ = 488 nm) and red filter (λ = 635 nm). Data was saved as listmode file (.LMD) then DNA ploidy and SPF were evaluated using the software Wincycle (Phoenix, USA), according to the recommendations. [11] DNA ploidy was defined by the DNA index (DI) which represents the ratio of tumor cells fluorescence to diploid control cells fluorescence: A tumor cell population with DI=1 is called DNA diploid, whereas it is called DNA aneuploid if at least one tumor cell population has a DI≠1. In addition to DNA diploid tumors, the S phase is called low (L) if SPF ≤ 1.2%, intermediate (I) if SPF > 1.2% and ≤ 3%, and high (H) if SPF is > 3%. For DNA aneuploid tumors, S phase is called low if SPF ≤ 3.85%, intermediate if SPF is > 3.85% and ≤ 8.7%, and high if SPF > 8.7%.

Statistical analysis

Averages, standard deviations and histograms were calculated using the Statview® , Abacus, Berkeley.


   Results Top


Quantification of centrosome

The centrosome appears as a round bright green fluorescent spot and all experiments were repeated five times (300 slides in total). A number of centrosomes higher than three was considered abnormal.

The majority of normal diploid mesothelial cells (94%) had a single fluorescent spot (centrosome) per cell [Figure 1]. In few cells (6%), two fluorescent spots were observed probably corresponding to replicating cells.
Figure 1: Immunostaining with anti-γ-tubulin observed under a Leica fluorescence microscope (×100), (a) mesothelial cells of ascites, (b) fibroblast cell line MRC5, (c) fibroblast cell line MRC5 in cycle, (d) breast cancer cell line MCF7, (e) breast cancer cell line T47D. Inset magnification (×400)

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When MRC5 diploid cells have reached 60% confluence, they have a high rate of mitoses and S-phase cells (30%) that allows studying the evolution of the centrosome during cell cycle by counting the number of centrosomes and by assessing the distance between two spots in the same cell. We observed 32% of cells with a single fluorescent spot and 68% of cells with two fluorescent spots [Figure 1].

The tumor cell lines MCF7 and T47D have one, two, three or four fluorescent spots [Figure 1]. Their number is detailed in [Table 1].
Table 1: Results for 10 samples of breast invasive carcinoma

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Ten samples of infiltrating ductal carcinoma of the breast were analyzed with the antibody anti-γ-tubuline [Figure 2]. The results of the count of centrosomes are shown in [Table 1].
Figure 2: (a) Cell prints of breast invasive ductal carcinoma (MGG, ×40); Immunostaining with anti-γ-tubulin observed under a Leica fluorescence microscope (×100), (b) one centrosome, (c) three centrosomes, (d) four centrosomes. Inset magnification (×400)

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All mesothelial cells were DNA diploid.

The MRC5 fibroblasts were DNA diploid with SPF depending on the rate of cell confluence: SPF was maximal (30%) at 60% confluence.

MCF7 and T47D cell lines were aneuploid with DI at 1.6 and 1.4 respectively.

Three cases of infiltrating ductal carcinoma were DNA diploid and the other seven cases were DNA aneuploid including three cases with DI around 1.5 (near triploid) and four cases with DI around 1.8 (near tetraploid). One single case of infiltrating ductal carcinoma had low SPF, two cases had intermediate SPF and five cases had high SPF.

Correlation between the number of centrosomes and DNA ploidy

Normal cells


In mesothelial cells (diploid cells with few or no cycles), the frequency of cells with one centrosome was 94%.

In MRC5 fibroblasts (diploid cells) with SPF of 30%, the percentage of cells with centrosome was 32% and the percentage of cells with two centrosomes was 68%.

These results correspond to what was expected in normal cells and therefore the quality control of experiments.

Tumor cell lines

In our study, 100% of MCF7 cells were DNA aneuploid with DI = 1.6. The percentage of cells with a number of centrosomes ≥ 3 was 64% in correlation with DNA aneuploidy.

Similarly, 100% of T47D cells were DNA aneuploid with DI = 1.4. The percentage of cells with a number of centrosomes ≥ 3 was 23%.

Invasive breast carcinoma

Three cases of invasive breast ductal carcinoma were DNA diploid and seven cases of invasive ductal breast carcinoma were DNA aneuploid. The percentage of cells with a number of centrosomes ≥ 3 was slightly higher in DNA aneuploid cases compared to DNA diploid cases (15% ± 9.6 vs. 13.3 ± 5.7%). A detailed analysis of correlations is shown in [Figure 3].
Figure 3: Correlation with the number of centrosomes in 10 cases of infiltrating ductal carcinoma based on: (a) DNA-ploidy (b) DNA index

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   Discussion Top


A century ago, Theodore Boveri had predicted that the slightest error during mitosis could lead to chromosomal instability and cancer. Because the centrosome plays an important role in cellular polarity and chromosomal segregation, cellular abnormalities such as increased numbers of centrosomes may increase the formation of multipolar spindles that lead to aneuploidy, which were frequently observed in many human cancers. [12],[13],[14],[15],[16],[17],[18],[19],[20] In addition, DNA ploidy is a good reflection of genomic instability and has been shown to be related to prognostic factors in breast cancer. [21],[22] However, a correlation between centrosome abnormalities and tumor aggressiveness has not been demonstrated yet.

Chromosomal abnormalities in cancer cells are frequently related to their structure or their number. The abnormal number called aneuploidy is a factor of poor prognosis. This may be an early event in relation with tumorigenesis or a late event in relation with aggressiveness of the tumor. Several recent studies converge both to understand these mechanisms and to identify new factors and tools for a better targeted therapy. Centrosomic amplification has been implicated in mitotic aberrations, aneuploidy and genomic instability. In humans, dysfunction of the centrosome would lead to a disruption of the mitotic spindle responsible for chromosomal abnormalities and DNA aneuploidy. [15] Recent studies exploring the relationship between aneuploidy of cancer cells, genomic instability and centrosomic aberrations have found cells with centrosomic abnormalities in an average of 9.6% of invasive carcinomas. [14] Moreover, some authors have reported the clinical value of DNA analysis by flow cytometry confirming the link between the fraction of cells in S phase, DNA ploidy and risk of relapse in patients with T1 or T2 breast cancer. [4],[5] The aim of our study was to find a relationship between abnormalities of the centrosome, DNA ploidy and SPF (DNA ploidy and SPF being a reflection of both genomic instability and cell proliferation). We explored the correlation between the number of centrosomes in the cell and DNA ploidy.

As control, we used normal cells theoretically having a single centrosome and cell cycles with one or two centrosomes. Mesothelial cells were normal diploid cells with the majority having a single centrosome. MRC5 diploid fibroblasts have a majority (68%) of cells with two centrosomes. These experiments were used to determine the normal diploid control cells. They allowed validating the technique because of the presence of only one or two centrosomes in each cell.

Tumor cell lines MCF7 and T47D cells are used as control for DNA aneuploidy. The analysis of MCF7 cells found 100% of DNA aneuploid cells with 64% having a number of centrosomes ≥ 3. These cells with DNA aneuploidy and DI close to 1.6 highly suggest a correlation between DNA aneuploidy and the number of centrosomes. The analysis of T47D cells showed a frequency of DNA aneuploid cells of 100% and DI close to 1.5 with a majority of cells with 1 or 2 centrosomes. Although the DI is close to 1.5, we found dissociation between DNA ploidy and the number of centrosomes. Initially, a karyotype should be performed with the aim of verifying the number of chromosomes in each line and verify whether the DNA aneuploidy observed corresponds to an increase in the number of chromosomes.

In the cases of invasive breast cancer, our findings failed to join the results observed by Lingle et al.[23] We did not observe a significant correlation between DNA ploidy, SPF and the number of centrosomes. In DNA diploid tumors, the frequency of cells with a number of centrosomes ≥ 3 was relatively low (13.3%). This result could be due to a low rate of aneuploid cells that were detected by FCM. In DNA aneuploid tumors, the rate of cells with abnormal number of centrosomes was slightly higher than in DNA-diploid tumors (15% vs 13.3%). Of the seven DNA aneuploid tumors, three tumors "near triploid" (DI around 1.5) had a low frequency of cells with three centrosomes, however one of these tumors had only 13.5% aneuploid cells. The four tumors "near tetraploid" did not have a significantly higher rate of cells with two or four centrosomes than other tumors. This lack of relationship may be due to the small number of cases in our study. It may also be due to tumor samples since the cells analyzed on the prints were different from those used for the FCM or to the fact that our analysis included the number and not the size or morphological abnormalities of the centrosome. Some difficulties may have also been encountered in the count of centrosomes in case of altered or small cells.

Centrosome abnormalities are considered markers of cellular carcinogenesis. Centrosomic amplification has been related to mitotic aberrations, aneuploidy and genomic instability in several cancerous lesions. These anomalies are also sought in breast cancer. Our pilot study allowed optimizing and validating a cytological method for identification and counting of centrosomes. Initial results showed no correlation between DNA aneuploidy and the number of centrosomes due to the small number of samples and the lack of study of morphological abnormalities of centrosomes. Further analysis of a larger number of samples and breast diseases, a greater number of cells per sample, as well as the study of centrosomal morphology is warranted.

 
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Correspondence Address:
Rita A Sakr
Department of Gynecology, Hopital Tenon, 4 rue de la chine, 75020 Paris
France
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9371.97150

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