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Comparative Study
. 2003 Jul 22:4:8.
doi: 10.1186/1471-2121-4-8.

Expression of cyclins A, E and topoisomerase II alpha correlates with centrosome amplification and genomic instability and influences the reliability of cytometric S-phase determination

Ulrike Kronenwett  1 , Juan Castro, Uwe J Roblick, Kaoru Fujioka, Carin Ostring, Farinaz Faridmoghaddam, Nongnit Laytragoon-Lewin, Bernhard Tribukait, Gert Auer
Affiliations
Comparative Study

Expression of cyclins A, E and topoisomerase II alpha correlates with centrosome amplification and genomic instability and influences the reliability of cytometric S-phase determination

Ulrike Kronenwett et al. BMC Cell Biol. .

Abstract

Background: The progression of normal cells through the cell cycle is meticulously regulated by checkpoints guaranteeing the exact replication of the genome during S-phase and its equal division at mitosis. A prerequisite for this achievement is synchronized DNA-replication and centrosome duplication. In this context the expression of cyclins A and E has been shown to play a principal role.

Results: Our results demonstrated a correlation between centrosome amplification, cell cycle fidelity and the level of mRNA and protein expression of cyclins A and E during the part of the cell cycle defined as G1-phase by means of DNA content based histogram analysis. It is shown that the normal diploid breast cell line HTB-125, the genomically relatively stable aneuploid breast cancer cell line MCF-7, and the genomically unstable aneuploid breast cancer cell line MDA-231 differ remarkably concerning both mRNA and protein expression of the two cyclins during G1-phase. In MDA-231 cells the expression of e.g. cyclin A mRNA was found to be ten times higher than in MCF-7 cells and about 500 times higher than in HTB-125 cells. Topoisomerase II alpha showed high mRNA expression in MDA compared to MCF-7 cells, but the difference in protein expression was small. Furthermore, we measured centrosome aberrations in 8.4% of the MDA-231 cells, and in only 1.3% of the more stable aneuploid cell line MCF-7. MDA cells showed 27% more incorporation of BrdU than reflected by S-phase determination with flow cytometric DNA content analysis, whereas these values were found to be of the same size in both HTB-125 and MCF-7 cells.

Conclusions: Our data indicate that the breast cancer cell lines MCF-7 and MDA-231, although both DNA-aneuploid, differ significantly regarding the degree of cell cycle disturbance and centrosome aberrations, which partly could explain the different genomic stability of the two cell lines. The results also question the reliability of cytometric DNA content based S-phase determination in genomically unstable tumor cell populations.

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Figures

Figure 1
Figure 1
Image cytometry Nuclear DNA content of the cell lines HTB-125, MCF-7 and MDA-231, exposed to serum starvation, and measured by image cytometry of Feulgen stained cells (2c denotes diploid DNA content). We measured 204 cells of the MDA-231 cell line, and 150 cells in MCF-7 and HTB-125 each. G1 indicates the percentage of cells in G1-phase of the cell cycle. S and G2 indicate the percentage of cells in S and G2-phase, respectively. G2 Exc is the percentage of cells with DNA content values exceeding twice the modal value plus 1c.
Figure 2
Figure 2
Real time qPCR and immunoassays Relative amount of cyclin A, E and topoisomerase II α mRNA determined by running real time quantitative PCR on reverse transcribed total RNA, extracted from HTB-125, MCF-7 and MDA-231 cells in G1-phase, which were assorted by centrifugal elutriation. The algorithm, chosen for calculating the relative amount of target mRNA gives values without any units of measurement. The detection range of the instrument is in our case five orders of magnitude. For the protein determination we calculated the percentage of positively stained G1-phase cells that were assorted by flow cytometry.
Figure 3
Figure 3
Flow cytometry and BrdU incorporation DNA histograms of HTB-125 (a), MCF-7 (c), MDA-231 (e) cell lines and two parameter histograms (BrdU incorporation vs. DNA content) of HTB-125 (b), MCF-7 (d) and MDA-231 (f) cell lines. As one can see the S-phases in HTB-125 and MCF-7 are very similar with both methods, but in the unstable aneuploid cell line MDA-231, there is a very substantial difference between the measurements with the two methods.
Figure 4
Figure 4
Centosome measurements Percentage of cells that show more than two centrosomes per cell in the cell lines HTB-125, MCF-7 and MDA-231, examined with a Zeiss Axioskop fluorescence microscope.
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