Paraffin-embedded tissue blocks from 130 gastric and/or esophageal resections due to upper gastrointestinal adenocarcinomas, performed between 1995 and 2005, were obtained from Vanderbilt University Medical Center (Nashville, Tenn) for immunohistochemical analysis. They included 44 lower esophageal, 43 gastroesophageal junction, 8 cardia, and 30 distal gastric (antrum and body) tumors. Tumor grading was performed according to World Health Organization (WHO) standards. DNA and mRNA purification were performed by using Qiagen purification kits (Qiagen, Hilden, Germany). Single-strand cDNA was synthesized by using the Advantage real-time polymerase chain reaction (RT-PCR Kit; Clontech, Palo Alto, Calif).
Quantitative real-time polymerase chain reaction (qRT-PCR)
mRNA was isolated from 25 normal gastric cardia samples and 45 primary upper gastrointestinal adenocarcinomas. Gene-specific primers for AURKA, PUMA, NOXA, and HPRT1 were designed, and the results were normalized to HPRT1 as described earlier.18 All primers were purchased from GeneLink (Hawthorne, NY), and their sequences are available from the authors upon request. qRT-PCR was performed by using an iCycler (Bio-Rad, Hercules, Calif) with a threshold cycle number determined with the use of iCycler software version 3.0. The reactions were performed in triplicate, and threshold cycle numbers were averaged. The fold change in all samples was calculated according to the formula 2(Rt−Et)/2(Rn−En), as described previously.7
Tissue microarrays and immunohistochemistry of AURKA protein
All tumor and normal gastric and esophageal mucosal epithelial tissues were histologically verified, and representative regions were selected for inclusion in a tissue microarray (TMA). The tumors were classified into intestinal and diffuse types19 and ranged from well differentiated (WD) to poorly differentiated (PD). Clinical staging was performed according to American Joint Committee on Cancer (AJCC) criteria.20 Tissue cores with a diameter of 0.5 mm were retrieved from the selected regions of the donor blocks and punched to the recipient block by using a manual tissue-array instrument (Beecher, Silver Spring, Md). Sections (5 μm) were transferred to polylysine-coated slides (SuperFrostPlus; Menzel-Glaser, Brunschwig, Germany) and incubated at 37°C for 2 hours. The resulting TMA was used for immunohistochemical analysis. An avidin-biotin immunoperoxidase assay was performed after pretreatment in a microwave with a citrate buffer for 20 minutes, and rabbit anti-AURKA (KR051; 1:100 dilutions; TransGenic, Kobe, Japan) was applied at room temperature. Immunohistochemical results were evaluated for intensity and staining frequency of nuclear and cytoplasmic components. The intensity of staining was graded 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The frequency was graded from 0 to 4 according to the percentage of positive cells as follows: 0, ≤3%; 1, ≤4% to 25%; 2, ≤26% to 50%; 3, ≤51% to 75%; 4, ≥75%. The products of multiplication of the intensity and frequency grades were then classified into an index core on a scale of 0 to 3: index score 0 = product of 0 (negative), index score 1 = products of 1 and 2 (weak), index score 2 = products of 3 and 4 (moderate), index score 3 = products of 6 through 12 (strong).
Cell culture, vectors, siRNA, and transfection
AGS, RKO, and RIE-1 cells were used in this study. Cells were cultured in F-12 (HAM) or Dulbecco modified eagle medium (DMEM) together with 10% fetal bovine serum (Invitrogen, Carlsbad, Calif) at 37°C in an atmosphere containing 5% CO2. AGS and RKO cells were obtained from American Tissue Culture Collection (ATCC, Manassas, Va). RIE-1 cells are spontaneously immortalized, nontransformed, and from an epidermal growth factor-responsive cell line (A generous gift from Dr. Robert. J. Coffey, Jr. at Vanderbilt University). The expression plasmid for AURKA was generated by polymerase chain reaction (PCR) amplification of the full-length coding sequence of AURKA and cloned in-frame into pcDNA3.1. A synthetic Flag tag sequence was added at the N-terminus of AURKA. Retroviral expression constructs, pBabe-puro, containing either the full-length of the AURKA coding sequence (pBabe puro-AURKA) or a kinase-dead AURKA mutant (D274A) were purchased from Addgene (Cambridge, Mass). AURKA-specific shRNA sequence (GATCCCC ATGCCCTGTCTTACTGTCATTCAAGAGATGACAGTAAGACAGGGCATTTTTTA) was cloned in retroviral expression construct pMSCV- siRNA-GFP by using Bgl II and Hind III restriction enzymes. A green fluorescent protein (GFP)-specific shRNA was cloned in the same vector and used as a negative control. In addition, we obtained validated small-interfering RNA (siRNA) oligonucleotides specific for AKT (#42,811) and a negative control siRNA (#4611) (Ambion, Austin, Tex). Transient transfections were performed by using Fugene 6 (Roche, Indianapolis, Ind) and Lipofectamine (Invitrogen, Carlsbad, Calif) following the manufacturers' protocols.
Ecopack 293 cells (5 × 105 per well) (Clontech, Palo Alto, Calif) were plated in a 6-well plate. Cells were transfected with retroviral vectors; empty pBabe-puro (control) and pBabe-puro-AURKA, respectively. After 48 hours of transfection, viruses were collected, filtered by using 0.45 μM filters, and added to RIE-1 cells in the presence of 8 μg/mL polybrene. The culture medium was replaced with fresh medium after 24 hours of transduction.
MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay
AURKA-overexpressing AGS cells (5 × 103 per well) were plated in a 96-well plate. The MTT Cell Assay kit from American Type Culture Collection (ATCC, Manassas, Va) was used to perform this assay as recommended by the manufacturer. Cells were plated in triplicate, and the assay carried out measurements for 3 days.
TUNEL (terminal deoxynucleotidyl transferase-mediated nick-end labeling) apoptosis assay
RKO cells were seeded into 8-well chamber slides and transfected with 400 ng of pcDNA3-flag-AURKA and pcDNA3 empty vector (control) per well. Cells were then treated either with camptothecin (5 μM) or dimethyl sulfoxide (DMSO; vehicle, control) for 24 hours. After 24 hours, DMSO-treated and camptothecin-treated cells were stained by terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) according to the manufacturer's instructions (Roche, Indianapolis, Ind), and then observed by tetramethylrhodamine isothiocyanate (TRITC) (red fluorescence). AURKA overexpression was determined by immunofluorescence staining in duplicate wells with the Flag antibody (Cell Signaling Technology, Boston, Mass) and fluorescein isothiocyanate (FITC)-goat anti-rabbit immunoglobulin G (IgG) (heavy + light [H + L]) conjugate (green; Jackson Immuno Research, West Grove, Pa). TUNEL-positive cells and AURKA-expressing cells (20 random fields at magnification of 40×, >400 cells) were then counted.
Cytochrome C release
AGS cells that stably expressed AURKA or pcDNA3 empty vector (control) were cultured into 8-well slide chambers (12 × 103 cell per well). Cells were then treated with DMSO (vehicle, control) or 20 μM camptothecin for 4 hours at 37°C. Cytochrome C release was detected by immunofluorescence by using mouse monoclonal anticytochrome-C antibody and by following the manufacturer's recommendations (Pharmingen, San Diego, Calif).
Fluorescence assorted cell sorting
Cells were trypsinized, washed twice with 1× ice-cool phosphate-buffered saline (PBS), and then resuspended in 0.2 mL ice-cold PBS, then fixed in 1mL ice-cool 70% ethanol, and incubated for 1 hour at −20°C. The cells were then centrifuged, resuspended in 1 mL PBS and treated with propidium iodide (50 μg/mL) and RNase (1 μg/mL) for 30 minutes at 37°C, and then analyzed by BD LSR II flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The data were analyzed by Becton Dickinson's BD FACS Diva software.
Western blot analysis
Cell lysates were prepared in a phosphate saline buffer (PBS) containing 1× protease cocktail inhibitor (Pierce, Rockford, Ill) and centrifuged at 3500 rpm for 10 minutes at 4°C. The protein concentration was measured by using a Bio-Rad protein assay (Bio-Rad, Hercules, Calif). Protein (10–15 μg) from each sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a nitrocellulose membrane. Target proteins were detected by using specific antibodies as follows: p53, HDM2, and p21Waf1 (Oncogene, San Diego, Calif); ACTIN, AKT, pAKTSer473, BAX, Anti-Flag, PUMA (Cell Signaling Technology, Boston, Mass); AURKA (TransGenic, Kobe, Japan); and NOXA (Imgenex, San Diego, Calif).
Luciferase activity assays were performed by using the pG13-Luc reporter plasmid, which contains 13 tandem repeats of the p53 consensus DNA-binding sites and measures the p53 transcription activity.21 The pcDNA3-p53 and pcDNA3-ΔNp73 are described elsewhere.22 PUMA-Luc luciferase vector was used to investigate the PUMA promoter activity. We determined the luciferase activity by using a Dual-Luciferase Reporter Assay kit (Promega, Madison, Wis). Results were normalized by using the Renilla luciferase activity (Renilla Luciferase Assay Lysis Buffer; Promega, Madison, Wis) or protein normalization as indicated. Results were averaged from 3 independent experiments and expressed as mean values with their individual data points.
For qRT-PCR data, mRNA expression levels between groups were assessed by using Kruskal-Wallis tests. When the overall effect was present, group-by-group comparisons were made while adjusting the significance level with the Scheffe method.23 Statistical comparisons were also performed by using the Student t test. P < .05 was considered to represent a statistically significant difference. For the immunohistochemical analysis results, patient characteristics and clinical variables were tabulated, and Fisher exact tests were used to compare these characteristics between the normal and the AURKA-overexpressed groups.