HSC-2, HSC-3, HSC-4, and Ca9-22 cell lines, derived from human OSCCs, were purchased from the Human Science Research Resources Bank, Osaka, Japan. H1 and Sa3 were kindly provided by Dr. S. Fujita of Wakayama Medical University, Wakayama, Japan. HNOKs were used as a normal control [22, 23]. All cells were grown in Dulbecco’s modified Eagle medium (DMEM) F-12 HAM (Sigma Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Sigma) and 50 units/ml penicillin and streptomycin (Sigma).
Primary OSCC samples and corresponding normal oral epithelial tissues were obtained at the time of surgeries performed at Chiba University Hospital. All patients provided informed consent for the study protocol, which was approved by the institutional review board of Chiba University. The tissues were divided into two parts, one of which was frozen immediately and stored at −80°C until RNA isolation, and the second was fixed in 20% buffered formaldehyde solution for pathologic diagnosis and IHC. The Department of Pathology, Chiba University Hospital, performed the histopathologic diagnosis of each tissue according to the World Health Organization criteria. Clinicopathological staging was determined according to the tumor-node-metastases classification of the International Union against Cancer. All OSCC samples were confirmed histologically and checked to ensure the presence of tumor in greater than 90% of specimens.
Preparation of cDNA
Total RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. cDNA was generated from 5 μg of total RNA using Ready-To-Go You-Prime First-Strand Beads (GE Healthcare, Buckinghamshire, UK) and oligo (dT) primer (Sigma Genosys, Ishikari, Japan), according to the manufacturer’s instructions.
mRNA expression analysis
qRT-PCR was performed to evaluate the expression levels of CDCA3 and Wee1 mRNA in OSCC-derived cell lines and HNOKs. We also evaluated the mRNA levels in primary OSCCs and paired specimens of normal oral tissues obtained from 69 patients. qRT-PCR was performed using LightCycler 480 apparatus (Roche Diagnostics GmbH, Mannheim, Germany). Primers were designed using the ProbeFinder qPCR assay design software, which is freely accessible at http://www.universalprobelibrary.com. The sequences of the gene-specific primers were as follows: CDCA3 forward 5’-TGGTATTGCACGGACACCTA-3’ and reverse 5’-TGTTTCACCAGTGGGCTTG-3’; Wee1 forward 5’-TTTGGTTCACATGGATATAAAACCT-3’ and reverse 5’-CCCAATCATCTTCGTCTCCT-3’. The PCR reactions were carried out in a final volume of 20 μl of a reaction mixture comprised of 10 μl of LightCycler 480 Probes Master (Roche), 0.2 μl of universal probe (Roche), and 4 μM of the primers, according to the manufacturer’s instructions. The reaction mixture was loaded onto the PCR plate and subjected to an initial denaturation at 95°C for 10 min, followed by 45 rounds of amplification at 95°C (10 sec) for denaturation, 60°C (30 sec) for annealing, and 72°C (1 sec) for extension, followed by a cooling step at 50°C for 30 seconds. The transcript amounts for the CDCA3 and Wee1 genes were estimated from the respective standard curves and normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward 5’-AGCCACATCGCTCAGACAC-3’ and reverse 5’-GCCCAATACGACCAAATCC-3’ transcript amount determined in corresponding samples.
Protein expression analysis
The cells were washed twice with cold phosphate buffered saline (PBS) and centrifuged briefly. The cell pellets were incubated at 4°C for 30 min in a lysis buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, and 10 mM Tris pH 7.4) with a proteinase inhibitor cocktail (Roche). The protein concentration was measured using the Bradford reagent (Bio-Rad, Richmond, CA). Protein extracts were electrophoresed on 4% to 12% Bis-Tris gel, transferred to nitrocellulose membranes (Invitrogen), and blocked for 1 hr at room temperature with Blocking One (Nacalai Tesque, Inc., Kyoto, Japan). The membranes were washed three times with 0.1 % Tween-20 in Tris-buffered saline and incubated with antibody for CDCA3 (Abcam, Cambridge, UK), p21Cip1, p27Kip1, p15INK4B, p16INK4A, CDK4, CDK6, Cyclin D1 (Cell Signaling Technology, Danvers, MA), and Cyclin E (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C and α-tubulin (Santa Cruz Biotechnology) 1 hr at room temperature. The membranes were washed again and incubated with a anti-rabbit or anti-mouse IgG (H + L) horseradish peroxidase conjugate (Promega, Madison, WI) as a secondary antibody for 1 hr at room temperature. Finally, the membranes were detected using SuperSignal West Pico Chemiluminescent substrate (Thermo, Rockford, IL), and immunoblotting was visualized by exposing the membranes to ATTO Light-Capture II (Tokyo, Japan). Signal intensities were quantitated using the CS Analyzer version 3.0 software (ATTO).
IHC of 4-μm sections of paraffin-embedded specimens was performed using rabbit anti-CDCA3 polyclonal antibody (Abcam). Briefly, after deparaffinization and hydration, the endogeneous peroxidase activity was quenched by a 30 min incubation in a mixture of 0.3% hydrogen peroxide solution in 100% methanol, after which the sections were blocked for 2 hr at room temperature with 1.5% blocking serum (Santa Cruz Biotechnology) in PBS before reaction overnight with anti-CDCA3 antibody (1:100 dilution) at 4°C in a moist chamber. Upon incubation with the primary antibody, the specimens were washed three times in PBS and treated with Envision reagent (DAKO, Carpinteria, CA) followed by color development in 3,3’-diaminobenzidine tetrahydrochloride (DAKO). The slides then were lightly counterstained with hematoxylin, dehydrated with ethanol, cleaned with xylene, and mounted. Non-specific binding of an antibody to proteins other than the antigen sometimes occurred. To avoid non-specific binding, an immunizing peptide blocking experiment was performed. As a negative control, triplicate sections were immunostained without exposure to primary antibodies, which confirmed the staining specificity. To quantify the status of the CDCA3 protein expression in those components, we used an IHC scoring system described previously [22–26]. This IHC scoring system was established for quantitative evaluation of IHC staining. The stained cells were determined in at least five random fields at 400 × magnification in each section. We counted 300 cells per one field of vision. The staining intensity (1, weak; 2, moderate; 3, intense) and the number of positive cells in the field of vision then were multiplied to calculate the IHC score using the following formula: IHC score = 1 × (number of weakly stained cells in the field) + 2 × (number of moderately stained cells in the field) + 3 × (number of intensely stained cells in the field). Cases with a CDCA3 IHC score exceeding 94.7 (maximum score within + 3 standard deviation (SD) of the mean of normal tissues) were defined as CDCA3-positive because 100% of the distribution falls within ± 3 SD of the mean in normal tissues. Two independent pathologists, both masked to the patients’ clinical status, made these judgments.
Stable transfection of CDCA3 shRNA
Stable transfection was performed at about 80% confluency in 24 well plates using Lipofectamine LTX and Plus Reagents (Invitrogen), according to the manufacturer’s instructions. Briefly, a total of 2 × 105 cells were seeded into each well in DMEM F-12 HAM (Sigma) containing 10% FBS (Sigma) without antibiotics. shCDCA3 and mock (0.1 μg) (Santa Cruz Biotechnology) vectors were transfected into OSCC-derived cells (H1 and Sa3) with 0.5 μl of Plus Reagents and 1.25 μl of Lipofectamine LTX. After transfection, the cells were isolated by the culture medium containing 2 μg/mL puromycin (Invitrogen). After 3 to 4 weeks, resistant cell clones were picked and transferred to 6-well plates and gradually expanded to 10-cm dishes. At 90% confluence, qRT-PCR and Western blot analyses were performed to assess the efficiency of CDCA3 knockdown.
To evaluate the effect of CDCA3 knockdown on cellular proliferation, we analyzed cellular growth in shCDCA3- and mock-transfected cells. These transfectants were seeded in 6-well plates at a density of 1 × 104 viable cells per well. The experiments were carried out for 168 hr, and the cells were counted every 24 hr. At the indicated time point, the cells were trypsinized and counted using a hemocytometer in triplicate samples.
To assess cell-cycle distribution of entire cell populations, the cells were harvested, washed with PBS, and probed with CycleTEST Plus DNA reagent kit (Becton-Dickinson, San Jose, CA), according to the manufacturer’s protocol. Briefly, the cells were centrifuged at 400 × g for 5 min. The cell pellets were resuspended with 250 μl of trypsin buffer, and incubated for 10 min at room temperature. We then added 200 μl of trypsin inhibitor and RNase buffer. Finally, the cells were labeled with 200 μl of propidium iodide stain solution. Flow cytometric determination of DNA content was analyzed by FACSCalibur (Becton-Dickinson). The fractions of the cells in the G0-G1, S, and G2-M phases were analyzed using Flow Jo software (Tree Star, Ashland, OR).
Statistical significance was determined using Fisher’s exact test or the Mann-Whitney’s U test. p < 0.05 was considered significant. The data are expressed as the mean ± standard error of the mean (SEM).