The study included as control a normal esophageal epithelial cell line (EPC-hTERT cells) [50, 51] as well as four esophageal cancer cell lines (OE21, Kyse-410, OE33, OE19). The esophageal cancer cell lines were originally derived from patients with esophageal squamous cell carcinomas (ESCC: OE21, Kyse-410), Barrett's adenocarcinoma (BAC: OE33) or an esophageal junctional adenocarcinoma (OE19) [47, 48]. Indeed, the specificity of the adenocarcinoma cell lines was recently approved . Due to clear adenocarcinoma differentiation and growth patterns, the two cell lines OE33, OE19 are collectively referred to as "BAC" in the present in vitro study, which does not address the carcinogenesis of esophageal carcinomas in view of the intestinal metaplasia-dysplasia-carcinoma sequence.
EPC-hTERT cells were cultivated in Keratinocyte-SFM medium (Invitrogen, Karlsruhe, Germany) supplemented with 40 μg/ml bovine pituitary extract (Invitrogen, Karlsruhe, Germany), 1.0 ng/ml EGF (Invitrogen, Karlsruhe, Germany), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Karlsruhe, Germany) at 37°C in a 5% CO2 atmosphere.
The esophageal cancer cell lines OE21 and Kyse-410 and the BAC cell lines OE33 and OE19 (European Collection of Cell Cultures, Salisbury, UK) were cultivated in RPMI 1640 medium (PAA Laboratories, Pasching, Austria), supplemented with 10% (v/v) Fetal Bovine Serum (PAA Laboratories, Pasching, Austria) and 2 mM GIBCO™ L-Glutamin (Invitrogen, Karlsruhe, Germany) at 37°C in a 5% CO2 atmosphere.
Hematoxylin and Eosin staining (HE staining)
Cells grown on coverslips were fixed with 4% paraformaldehyde (PFA), rinsed with Phosphate buffered saline (PBS: 137 mM NaCl, 10 mM Na2HPO4, 1.8 mM KH2PO4; pH 6.8-7) and stained with Hematoxylin (Carl Roth, Karlsruhe, Germany). After removing the hematoxylin solution mains water was added twice. Cells were stained with Eosin Y solution (Carl Roth, Karlsruhe, Germany) and distilled water was added. The coverslips were then immersed in an ascending ethanol series and in xylol.
Cell cycle phase distribution analysis by flow cytometry
For cell cycle distribution analyses by flow cytometry cells were grown to 50%-60% confluency. The cells in the medium and trypsinized cells were collected and fixed in ice-cold 70% (v/v) ethanol. After washing with PBS cells were stained with propidium iodide (20 μg/ml propidium iodide (Sigma-Aldrich, Steinheim, Germany), 0.1% (v/v) Tritron X-100, 0.2 mg/ml Ribonuclease A (Sigma-Aldrich, Steinheim, Germany) in PBS). Stained cells were analyzed using the LSRII system and DB FACS Diva software (Becton Dickinson, Heidelberg, Germany).
Fluorescence in situ hybridization (FISH)
Cells were grown on Poly-L-Lysine coated Lab-Tek® 1 Well Glass Slides (Thermo Fisher Scientific, Langenselbold, Germany). Cells were washed with PBS, fixed in 3:1 methanol/glacial acetic acid and dehydrated in an ethanol series. AURKA (20q13) & 20q11 DNA probe (Kreatech Diagnostics, Amsterdam, Netherlands) or AURKB (17p13)/Alphasatellite 17 specific DNA probe (MP Biomedicals, Illkirch, France) was applied. Co-denaturation was performed for 5 min at 75°C for AURKA or 80°C for AURKB probes and hybridization for 16-18 h at 37°C in a humidified chamber. After washing in 0.4× SSC/0.3% (v/v) NP-40 pH 7 for 2 min at 73°C and in 2× SSC/0.1% (v/v) NP-40 pH 7-7.5 for 1 min at room temperature (RT), cell nuclei were counterstained with DAPI (Vector Laboratories, Burlingame, USA). Examination was done at a fluorescence microscope (Axioplan2 imaging microscope equipped with a Plan-Apochromat 63×/1.4 oil objective, Carl Zeiss MicroImaging, Göttingen, Germany) with slider module. Image stacks at 0.9 μm intervals were taken of at least three representative fields per cell line. Image stacks were converted into 3D view by AxioVision software (Carl Zeiss MicroImaging, Göttingen, Germany).
For each cell line, the gene (AURKA or AURKB) and chromosome specific signals (centromer enumeration probes/CEP; CEP20 or CEP17) were counted per individual cell nucleus (range of cell nuclei counted: 49-88; a mean of 71.3 ± 13.1 cell nuclei per cell line). The mean and standard deviation of the gene (AURKA or AURKB) and chromosome specific signals (CEP20 or CEP17) of counted cell nuclei were calculated for each cell line. The FISH ratio (AURKA to CEP20 and AURKB to CEP17) was calculated for each analyzed cell nucleus and thereof the mean and standard deviation was calculated for each cell line. True gene-specific amplification was considered at a FISH ratio of >2. The FISH procedure and quantification has previously been published by us for evaluation of Aurora-A and other gene copy numbers in tissue specimens .
Indirect immunofluorescence and evaluation of mitoses
Cells were grown on coverslips, fixed in 2% PFA, washed in PBS and permeabilized in 0.5% (v/v) Tritron X-100 in PBS. After PBS washing, cells were incubated with blocking buffer (PBS containing 5.0% (v/v) normal goat serum and 0.3% (v/v) Tritron X-100). Diluted primary antibodies (mouse anti-IAK1/Aurora-A Kinase, 1:100, clone 4, BD Biosciences, Heidelberg, Germany; mouse anti-pericentrin, 1:1000, clone mAbcam 28144, Abcam, Cambridge, UK; mouse anti-p53, 1:50, clone DO-7, DakoCytomation, Hamburg, Germany) were incubated over night at 4°C, cells were rinsed with PBS and 1:200 diluted fluorescently labelled secondary antibodies (goat-anti-mouse IgG-Alexa488, Invitrogen, Karlsruhe, Germany), were incubated for 1 h at RT. After washing with PBS and distilled water, cell nuclei were counterstained with DAPI (Vector Laboratories, Burlingame, USA). Note that the p53 antibody used was raised against the N-terminal domain (amino acids 1-45), recognizing also mutated and expressed (also truncated) p53 proteins.
Normal bipolar mitoses were defined as mitotic cells with 2 Aurora-A positive centrosomes/spindle poles. Multipolar mitoses were defined as mitotic cells with >2 Aurora-A positive centrosomes/spindle poles. In three independent experiments, cells were screened using a x40 objective and a minimum of 100 cells were counted for the mitotic index (mitoses per cells counted in %; range of total cells counted: 100-124) and up to 100 mitoses per cell line were evaluated for the occurrence of multipolar mitoses (multipolar mitoses per mitoses counted; range of total mitoses counted: 81-104) (Table 2).
Preparation of total protein and determination of protein concentration was performed using the Qproteome™ Mammalian Protein Prep Kit (Qiagen, Hilden, Germany) and the DC Protein Assay (Bio-Rad, München, Germany) according to the manufacturer's protocols. 10 μg of total protein extracts per lane were loaded onto 10% polyacrylamide gels. Proteins were transferred onto Protran® Nitrocellulose Transfer Membrane (Whatman, Dassel, Germany) by Semi-Dry Blot. After blocking the membrane in 5% (m/v) nonfat dried milk powder in Tris buffered saline with Tween (TBST: 10 mM Tris-Base, 150 mM NaCl, 0.1% (m/v) Tween; pH 7.2-7.4), the primary antibodies diluted in 5% (m/v) nonfat dried milk powder in TBST (mouse anti-IAK1/Aurora-A Kinase, 1:250, clone 4, BD Biosciences, Heidelberg, Germany; rabbit anti-Aurora-B, 1:5000, clone EP1009Y. Epitomics, Burlingame CA, USA; mouse anti-p53, 1:1000, clone DO-7, DakoCytomation, Hamburg, Germany; mouse anti-β-Actin, 1:1000, clone AC-15, Sigma-Aldrich, Steinheim, Germany) or 3% BSA in TBST (rabbit anti Aurora-A/phospho-T288, 1:1000, clone C39D8, Cell signalling, Danvers MA, USA) or 5% BSA in TBST (rabbit anti-Aurora-B/phospho-T232, 1:500, Abcam, Cambridge, UK) were incubated. After HRP conjugated secondary antibody (1:25000, Dianova, Hamburg, Germany) incubation, the membrane was incubated with ECL reagents (GE Healthcare, Freiburg i. Br., Germany) and exposed to autoradiography films. Note that the p53 antibody used was raised against the N-terminal domain (amino acids 1-45), recognizing also mutated and expressed (also truncated) p53 proteins.
p53 mutation analysis
Genomic DNA was isolated using the QIAamp® DNA Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction. Amplification of p53 exons 2-11 was performed using primers and protocols slightly modified from previous studies [54, 55]. PCR was carried out in a 25 μl reaction mixture containing 1× PCR Buffer, 1.5-2.5 mM MgCl2, 12 ng/μl gDNA, 0.4 mM dNTP Mix, 0.4 μM forward and reverse primers and 1.25 U Taq DNA polymerase. The PCR was performed with the following conditions: 94°C for 4 min, 40 cycles consisting of 94°C for 30 sec, 53-65°C for 30 sec and 72°C for 30 sec, followed by 72°C for 7 min. PCR products were purified using the QIAquick® PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Sequencing was performed using BigDye® Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Darmstadt, Germany) according to the manufacturer's instruction. The reactions were performed in 20 μl reaction mixture consisting of 3-5 ng PCR product, 0.16 μM forward or reverse primers, 20% (v/v) BigDye® Ready Reaction Mix and 1× Big Dye® Sequencing Buffer. A positive control with a 20 μl reaction mixture containing 5% (v/v) pGEM®-3Zf(+) double-stranded DNA control Template, 5% (v/v) -21 M13 Control Primer (forward), 20% (v/v) BigDye® Ready Reaction Mix and 1× Big Dye® Sequencing Buffer was included. The PCR was performed with the following conditions: 96°C for 1 min, 24 cycles consisting of 96°C for 10 sec, 50°C for 5 sec and 60°C for 4 min. DNA was precipitated with ethanol containing 5 mM EDTA and 120 mM sodium acetate, dissolved in formamide and denatured for 5 min at 95°C. Capillary electrophoresis was performed using the ABI PRISM™ 310 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany). The Sequencing Analysis Software V 5.2 (Applied Biosystems, Darmstadt, Germany) was used to analyze the collected electropherogram traces and sequencing information. The p53 sequence of the GenBank database with accession number NC_000017.9|NC_000017:c7531642-7512445 was used as reference.
RNA isolation and cDNA synthesis
Total RNA isolation was performed using the RNeasy® Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction. For cDNA synthesis, a 9 μl reaction mixture containing 200 ng total RNA, 1 μl yeast RNA (10 ng/μl) and 2 μl Hexanucleotide Mix (10×, Roche Diagnostics, Mannheim, Germany) was incubated for 2 min at 70°C and 10 min at RT. A second 11 μl reaction mixture containing 4 μl First Strand Buffer (5×, Invitrogen, Karlsruhe, Germany), 2 μl DTT (0.1 M), 1 μl dNTP Mix (10 mM) and 1 μl M-MLV RT (200U/μl, Invitrogen, Karlsruhe, Germany), was added and incubated for 1 h at 37°C. The M-MLV RT was inactivated for 5 min at 95°C. For reverse transcription of Universal Human Reference RNA (uRNA) (Stratagene, Heidelberg, Germany) , the calibrator of qRT-PCR, 300 ng RNA was employed in an appropriate volume.
Quantitative reverse transcription PCR (qRT-PCR)
qRT-PCR was performed using established protocols for Aurora-A and TBP . Following Aurora-B primers and probes were used (sequence 5'-3'): Aurora-B-forward (900 nM): CAT GAG CCG CTC CAA TGT C, Aurora-B-reverse (50 nM): CCC AAT CTC AAA GTC ATC AAT TGT, Aurora-B-probe (150 nM): 6-FAM-ACA CCC GAC ATC TTA ACG CGG CA-TAMRA. The comparative Ct method was used to calculate Aurora-A/-B mRNA levels. The amount of the target gene is normalized to the endogenous housekeeping gene TATA box binding protein (TBP) and these relative fold differences are compared between the experimental and the uRNA calibrator sample [69, 70].