Cell lines and culture conditions
The DNA-diploid Hs 578Bst cell line (ATCC-HTB-125) was derived from normal human breast tissue peripheral to an infiltrating ductal carcinoma. The cells were grown in modified Dulbecco's medium with 30 ng/ml EGF (90%), 50 μg/ml penicillin, 50 μg/ml streptomycin, supplemented with 10% FBS.
The DNA-hypotetraploid MCF-7 cell line (ATCC-HTB-22) originally came from a human breast adenocarcinoma. The cells were grown in minimum essential medium (Gibco BRL) with 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 50 μg/ml streptomycin and 10% FBS.
The DNA-hypotriploid MDA-MB-231 cell line (ATCC-HTB-26) was derived from a human breast adenocarcinoma. These cells were grown in Dulbecco's MOD eagle medium (Gibco BRL) with 2 mM L-glutamine, 1.0 mM sodium pyruvate, 50 μg/ml penicillin, 50 μg/ml streptomycin, and 10% FBS.
The cells were incubated at 37°C in a humid atmosphere of 5% CO2 in 325 cm2 flasks and were subcultured when having reached confluence, using 0.25% trypsin-0.03% EDTA solution (Gibco BRL). For experimental purposes, cells were seeded at a concentration 10–20,000 cells/cm2 and harvested before having reached confluence. The medium was removed and the cells were then washed once in 0.25% trypsin (Gibco). After removing trypsin the cells were kept at room temperature for 10 minutes. Then they were shaken vigorously, 10 ml of PBS was added and the mixture was shaken again. The cell suspension was transferred to a 15 ml tube. After 5 minutes of centrifugation at 3,000 RPM, the supernatant was removed and the cells kept at 4°C until further treatment. Serum starvation was performed by culturing the cells in medium containing 0.1% FBS during 72 hours.
Separation of cells on the basis of different cell size was made in a Beckman JE-G centrifugal elutriator rotor. Exponentially growing cells were harvested by trypsination, washed, and resuspended in respective medium. Approximately 2 × 108 cells were elutriated in each run at 4°C. The rotor speed was 2,780 rpm, and the initial flow rate was 12 ml/min. Five fractions were obtained by stepwise increase of the flow rate up to 45 ml/min. The first fraction containing partly non-viable cells was discarded. The cells of the second fraction containing entirely G1 cells were collected by centrifugation and resuspended in PBS. Since about 4–6 × 106 cells were needed for the mRNA measurement, fractions from several elutriation runs were stored in PBS at -20°C. Aliquots were fixed in buffered formalin for flow-cytometric analysis of the cellular DNA content as described earlier .
Determination of mRNA
G1 cells assorted by centrifugal elutriation were used for RNA extraction, cDNA synthesis and real time quantitative PCR with an amplicon for cyclins A, E and topoisomerase II α.
RNA extraction and DNase treatment
Total RNA was extracted using the Trizol reagent, followed by treatment with DNAse I (both reagents from Life Technologies, Paisley, UK). After 15 minutes of incubation at room temperature, the enzyme was inactivated with 25 mM EDTA, followed by heating for 10 minutes at 70°C to denature DNase.
We reverse transcribed 2 μg of total RNA in a final reaction volume of 20 μl, containing 1xFirst Strand Buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2), 100 ng random hexamers, 0.1 M DTT, 10 mM dNTP mix, Applied Biosystems (ABI), 200 units Superscript II RNase H- Reverse Transcriptase and DEPC treated water (all other reagents from Life Technologies). First, the mixture of total RNA, random hexamers and DEPC treated water was kept at 70°C for 10 minutes and afterwards put on ice. Then the mix containing buffer, DTT and dNTPs was added, followed by incubation at 25°C for 10 minutes. After two minutes at 42°C, the Superscript II was spiked in for the remaining 50 minutes of reverse transcription. We stopped the reaction by heating to 70°C for 15 minutes.
Real time quantitative PCR
With real time PCR the measurement is taking place at a quite early stage during the polymerase chain reaction. At the exponential phase of the amplification process, all reagents are still abundant and there is no danger of limiting conditions, which might interfere with a precise quantitation. This is an advantage over endpoint analysis, where small variations in starting reaction conditions may escalate and lead to totally different final product yield for the same starting copy number of template . Concerning real time quantitation the crucial value is the threshold cycle (CT), which is the fractional cycle number during PCR at which the amount of amplified target in each sample reaches a fixed threshold above the background noise [35, 36]. The product accumulation is directly proportional to the increase in fluorescent emission that occurs if the Taq polymerase is performing a 5'-3' nucleolytic digestion of the dual-labeled fluorogenic probe, which has to be hybridized to the template. On the other hand, the CT-value is inversely proportional to the logarithm of the initial template copy number [37–39]. As long as the 5'-reporter dye (distinct fluoresceins: FAM, VIC, TET) is in a certain spatial proximity to the 3'-quencher dye (TAMRA, 6-carboxy-tetramethylrhodamine), no fluorescence is detectable .
Relative quantitation of cyclin A, E and topoisomerase II α mRNA levels
Cyclin A, E and topoisomerase II α mRNA levels were determined by performing real time PCR. We did a relative quantitation according to the comparative CT method, 18S rRNA served as the endogenous external control and our inter-run standard was cDNA prepared from asynchronous MDA-MB-231 cells. All PCR-reactions were performed on a 7700 Sequence Detector (ABI). We ran each amplicon (cyclins A, E, topoisomerase II α and 18S rRNA) in triplicates for all cell lines. From the mean CT-values of the respective triplicates the mean CT-value of the 18S rRNA amplicon was subtracted. The purpose of the endogenous control is to correct for differences in pipetting the cDNA from the cell lines and to compensate for putative reverse transcription and PCR inhibition. This subtraction gave us ΔCT-values. The CT-value of the inter-run standard was subtracted from all the other ΔCT-values, and that resulted in ΔΔCT-values. The last step in getting the relative amount of target mRNA is to calculate 2-ΔΔCT .
Primers, probes and the PCR reaction
Primers and probes for the targets cyclin A, E and topoisomerase II α were found with the aid of Primer Express 1.0 (ABI, Foster City, CA), a computer program specialized in designing short amplicons for real time PCR. To exclude an amplification from genomic DNA, we placed one primer of each amplicon across an exon/exon junction in getting not more than six bases of the 3'-end on one exon and the remaining nucleotides of this primer on the adjoining one. The probes we purchased from ABI, Warrington, Cheshire, UK; primers were ordered from Life Technologies. The complete 18S rRNA amplicon was already designed by ABI.
The reaction volume for the PCR was 25 μl. Each master mix comprised 1xTaqMan buffer A, 6 mM MgCl2, 200 μM dATP, dCTP, dGTP, dTTP and 0.625 units of AmpliTaq Gold DNA polymerase (all reagents from ABI). We used the probe for cyclins A, E and topoisomerase II α at a concentration of 100 nM and the probe for 18S rRNA at a concentration of 75 nM. The primers for cyclins A, E and topoisomerase II α had the concentration of 200 nM, in the case of the 18S rRNA the concentration of the forward primer was 50 nM, those of the reverse was 40 nM. Per 25 μl reaction volume 25 ng reverse transcribed total RNA was added. The thermal cycling conditions were as follows: 10 minutes at 95°C (activation of the Taq polymerase), and 40 cycles at 95°C for 15 seconds and 62°C for 1 minute. All target genes and the endogenous control were run in triplicates for all cell lines. Additionally we ran non-template controls in triplicates for all targets and the endogenous control.
Image cytometry was performed on Feulgen stained cells. The staining and standardization were based on methods previously described .
A cell line was defined as genomically relatively stable provided that it showed a) confluence and/or serum starvation induced growth arrest in G0/G1, b) low percentage of cells with nonmodal DNA content values (Fig. 1). Consequently, a genomically unstable cell line was defined by a) unchanged high proliferative activity at confluence and/or upon serum starvation, b) high percentage of cells with nonmodal DNA content values (Fig. 1).
Single parameter DNA flow cytometry
For DNA histograms, cells were harvested and analysed as described previously . Briefly, cells were fixed in 4% buffered formaldehyde for 18 hours at room temperature. Formaldehyde was removed by 95% ethanol for 1 hour followed by rehydration in distilled water for 1 hour. After treatment with subtilisin Carlsberg solution [0.1% Sigma protease XXIV, 0.1 M Tris and 0.07 M NaCl (pH 7,5)] and staining with DAPI-Sulforhodamine solution [8 μM DAPI, 50 μM Sulforhodamine 101, 0.1 M Tris and 0.07 M NaCl (pH 7.5)], samples were analysed using a PAS II flowcytometer (Partec, Münster, Germany) equipped with a 100 W mercury arc lamp HBO 100. DAPI fluorescence was measured above 435 nm. The multicycle program for cell cycle analysis (Phoenix flow systems, San Diego, CA) was used for histogram analysis; the number of nuclei/histogram was 40,000. The S-phase was fitted to a broadened trapezoidal model. For background correction the sliced-nuclei model was applied.
Two parameter DNA-BrdU analysis
For BrdU (5'-Bromo-2'-deoxyuridine) labeling, cells were precultured for 24 hours, pulse labeled for 10 minutes with 20 μM BrdU, washed twice with culturing medium and then recultured with new medium for 20 minutes, followed by harvesting as before and fixation in 4% buffered formaldehyde. Immunostaining was carried out using a modified technique described by Sangfelt et al.  with 2 mg/ml monoclonal anti-BrdU antibodies (Boehringer Mannheim Scandinavia AB, Bromma, Sweden) for 1 hour at room temperature and FITC-conjugated anti mouse IgG antibodies (F-0257, Sigma), diluted 1/100 in PBT (PBS, 2% BSA, 0,5% Tween 20) for 1 hour in the dark at room temperature. DNA was stained with 10 μM propidium iodide (PI) for 20 minutes. A Nikon fluorescence microscope equipped with a mercury arc lamp HBO 100 was used for control of the quality of immunostaining. The samples were analysed in a FACScalibur flowcytometer (Becton Dickinson, Sweden) for two-parameter dot plot histogram analysis (BrdU incorporation vs. DNA content).
Cell staining and indirect immunofluorescence
Indirect immunofluorescence was performed by the method of Gong et al. . As negative control, we used FITC-conjugated mouse IgE monoclonal antibody (13834X, Pharmingen). All four antibodies: cyclin E (HE 121, Santa Cruz Biotechnology), cyclin A (6E6, Novocastra Laboratories UK), topoisomerase II α (Novocastra Laboratories UK) and IgE were incubated for 1 hour at room temperature, followed by incubation with the secondary antibody (FITC-conjugated IgG anti mouse antibody) for 1 hour in the dark at room temperature diluted 1/100 in PBT. DNA was stained with propidium iodide (10 μg/ml in PBS+RNAse 1 mg/ml). The samples were kept in the dark for 20 minutes before running them in the FACS. The FACS was equipped with an argon laser; the FITC fluorescence was analysed at 515 nm and the PI at 488 nm. 20,000 events were stored for every sample. The Cell Quest (Becton Dickinson) program was used for the two-parameter dot plot histogram analysis.
Cells were seeded onto sterile glass slides in culture dishes and cultivated for 3 days. After rinsing with PBS, cells were fixed in methanol at -20°C for 10 min and in acetone at -20°C for 6 min. After each incubation cells were extensively rinsed with PBS. All procedures were done at room temperature. Blocking solution (1% normal goat serum, 0.1% Tween 20/PBS) was applied to the cells for 30 min at 37°C. Cells were incubated with anti γ-tubulin antibody (Sigma T6557), diluted 1:1000 in PBS, containing 2% normal goat serum, for 1 hour. Antibody-antigen complexes were detected by FITC-conjugated anti mouse IgG. Stained slides were mounted with Vectashield mounting medium. With a Zeiss Axioskop fluorescence microscope, the proportion of cells with more than two centrosomes was counted in more than 500 cells of each cell line.