Two human CRC cell lines, SW480 and HCT-8, exhibit different expression levels of B7-H3. We constructed SW480 cells that expressed high levels of B7-H3 (SW480-B7-H3) and HCT-8 cells that were stably transfected with B7-H3 shRNA (HCT-8-shB7-H3). Cells transfected with a mock vector were used as negative controls (SW480-NC and HCT-8-NC). Cells were maintained in Dulbecco's modified Eagle's medium or RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS), at 37°C in a humidified atmosphere with 5% CO₂.

5-fluorouracil (5-Fu) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-human B7-H3, β-actin and BRCC3 antibodies were purchased from Abcam (Cambridge, MA, USA); the α-tubulin antibody was purchased from Biotime (Shanghai, China); the horseradish peroxidase-conjugated secondary anti-rabbit antibodies were purchased from Beyotime Institute of Biotechnology (Nantong, China); the secondary antibodies fluorophore-labeled donkey anti-rabbit IgG (A11374) were purchased from Invitrogen (Carlsbad, CA, USA).

SW480-NC/SW480-B7-H3 or HCT-8-NC/HCT-8-shB7-H3 cells were seeded at 3,000 cells/well in 96-well plates in triplicate. The next day, the medium was replaced with medium containing 5-Fu at a 2-fold concentration gradient. After 48 h, 10 µl CCK-8 was added to each well, and the absorbance was recorded on a BioTek microplate reader at the wavelength of 450 nm. Cell viability was assessed in percent wherein vehicle-treated cells were taken as 100%. Half maximal inhibitory concentration (IC₅₀) was calculated using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA).

Total RNA was isolated from 1.5×10⁶ cells using TRIzol, following the manufacturer's instructions, and then treated with RNase-free DNase to remove residual genomic DNA. The first strand cDNA was synthesized from 1 µg RNA using a SuperScript First-Strand Synthesis system (Invitrogen). First-strand cDNA was amplified in a 20 µl PCR reaction mixture: 10 µl 2X SYBR-Green PCR Master Mix, 0.4 µl 50X ROX, 0.4 µl of each specific primer set and ddH₂O added to 20 µl. The sequences of primers were as follows: β-actin 5′-AGCGAGCATCCCCCAAAGTT-3′ (sense) and 5′-GGGCACGAAGGCTCATCATT-3′ (antisense); BRCC3 5′-AGAGTTCAGAGTATGAGAGAATCG-3′ (sense) and 5′-TCTTGGTTACTGAGTCCAGATGT-3′ (antisense); The PCR cycling consisted of 40 cycles of amplification of the cDNA with annealing at 60°C.

Western blot assays were performed on whole-cell extracts prepared by lysing 1×10⁶ cells containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and PMSF protease inhibitor. Equal protein loading of the lysates was achieved by standardization with the BCA protein assay kit from Biotime. Samples were separated on SDS-PAGE gels and transferred to nitrocellulose transfer membranes. After blocking with 5% skim milk in TBST, the membranes were incubated with the appropriate primary antibody (α-tubulin or BRCC3) overnight at 4°C, followed by incubation with HRP-conjugated secondary antibody for 2 h at room temperature. The protein bands were visualized with an enhanced chemiluminescence.

Cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 10 min at room temperature (RT). Cells were washed three times in PBS after each treatment. After blocking with 2% BSA in PBS for 30 min at room temperature, the cells were incubated with primary antibody in 2% BSA/PBS overnight at 4°C and then incubated with fluorescent secondary antibody for 1 h at room temperature. Cell nuclei were stained with DAPI for 10 min at RT. Images were acquired with the TSC SP8 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany).

The comet assay was performed under alkaline conditions according to Singth et al (14). Cells were seeded in 12-well tissue-culture plates and incubated for 24 h for cell attachment. Subsequently, cells were treated with increasing concentrations of 5-Fu (positive control) for 24 h. Cells were harvested, washed and resuspended in ice-cold PBS. Approximately 25 µl of the resuspended cells were mixed with 75 µl of low melting point agarose at 37°C; the suspension was spread over the well with the pipette tip. The slides were placed at 4°C in the dark until gelling occurred and then were immersed in pre-chilled lysis buffer at 4°C. After incubating for 2 h, the buffer was aspirated and replaced with pre-chilled alkaline solution for 30 min at 4°C. After lysis and unwinding, the slides were placed in a horizontal electrophoresis tank filled with freshly prepared alkaline electrophoresis buffer. The electrophoresis was run at 20 V and 200 mA for 30 min. After electrophoresis, the slides were transferred to pre-chilled distilled water for 2 min, then aspirated and repeated twice. The final rinse was aspirated and replaced with cold 70% ethanol for 5 min. Thereafter, the slides were allowed to air dry, and 100 µl/well of PI was added to each slide for 20 min in the dark at room temperature for DNA staining. DNA migration was observed using a Nikon 90i fluorescence microscope. For each concentration, 6–10 randomly selected cells were analyzed. All of the comet images were analyzed using CASP software (CASPlab, Wroclaw, Poland) (15), and the percentage of DNA in the comet tail (TDNA%), tail length (TL), tail moment (TM) and olive tail moment (OTM) were recorded to characterize the cell DNA damage. All the experiments were repeated once, and the variation between experiments was analyzed.

Colony formation assays were performed by replating cells at a density of 300 cells/well onto a 6-well culture plate in medium containing 10% FBS. After 3 weeks, the cells were fixed with ice-cold methanol for 10 min and then stained by using 0.05% crystal violet in ddH₂O for 15 min. Alternatively, for examination of clonogenic ability of SW480 and HCT-8 cells with 5-Fu treatment, a density of 600 cells/well were seeded onto a 6-well culture plate in medium containing 10% FBS and 2 µg/ml 5-Fu or vehicle was added to the cultures at day 3 after seeding. The cultures were continuously maintained for another 2 weeks and subjected to the colony formation assay. Colonies produced by each cell-group were counted and measured using ImageJ software.

siRNA against BRCC3 were chemically synthesized by RiboBio Co., Ltd. (Guangzhou, China). Transfection of siRNAs was performed using the Oligofectamine reagent (Invitrogen). The sequences of siRNA-BRCC3 are as follows: siRNA1-GCAGGAATTACAACAAGAA, siRNA2-GAAGGACCGAGTAGAAATT.

All experiments were re-performed at least three times with similar results. Quantitative data were presented as the average value of replicates ± SD (standard deviation) within the representative experiment.

To inhibit tumor growth, we incubated SW480-NC, SW480-B7-H3, HCT-8-NC, or HCT-8-shB7-H3 cells with a concentration gradient of 5-Fu for 48 h (Fig. 1A). CCK-8 assays showed that the inhibition rate of SW480-B7-H3 was less than SW480-NC at any concentration of 5-Fu (P<0.05). The IC₅₀ of 5-Fu increased from 10.58 to 32.46 µg/ml after B7-H3 was upregulated. The HCT-8 pairs showed similar results (P<0.05): the IC₅₀ of 5-Fu decreased from 31.28 to 14.87 µg/ml after B7-H3 was knocked down. We next studied the ability of cancer cells to form colonies on 6-well plates in presence or absence of 5-Fu. Consistent with the CCK-8 assay results, the colony formation of cells overexpressing B7-H3 (SW480-B7-H3 and HCT-8-NC) was increased compared with the cells with low expression of B7-H3 (SW480-NC and HCT-8-shB7-H3) (Fig. 1B). Therefore, these observations demonstrate that the overexpression of B7-H3 weakened the sensitivity of CRC cells to 5-Fu.

The mechanism by which B7-H3 induces resis tance to 5-Fu in cancer cells is very complicated. We have previously reported that B7-H3 could enhance cancer cell resistance to apoptosis by increasing the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl. Here, we examined the expression of B7-H3 in stably transfected CRC cells using western blot analysis (Fig. 2A) and analyzed BRCC3 expression in response to B7-H3 overexpression or knockdown using real-time PCR (Fig. 2B), western blot analysis (Fig. 2C) and immunofluorescence (Fig. 2D). Both mRNA and protein levels of BRCC3 in SW480-NC, SW480-B7-H3, HCT-8-NC, and HCT-8-shB7-H3 cells corresponded with B7-H3 expression, such that B7-H3-overexpressing cells (SW480-B7-H3 and HCT-8-NC) demonstrated higher BRCC3 expression compared to cells having low B7-H3 expression (SW480-NC and HCT-8-shB7-H3). Immunofluorescence showed that BRCC3 expression was predominantly cytosolic.

Since BRCC3 has been reported to be involved in mediating DNA repair (16), we examined its role in repairing 5-Fu-induced DNA damage in CRC cells (Fig. 3). SW480-NC and SW480-B7-H3 cells were treated with 5-Fu (0, 12.5 and 25 µg/ml; Fig. 3A), and HCT-8-NC and HCT-8-shB7-H3 cells were treated with 5-Fu (0, 25 and 50 µg/ml; Fig. 3B) for 24 h; subsequent DNA damage was measured by DNA comet assay. As depicted, treatment with 12.5 or 25 µg/ml 5-Fu induced longer DNA tails and higher tail-DNA content in SW480-NC versus SW480-B7-H3 cells, indicating greater DNA damage in cells expressing less B7-H3. Similarly, treatment with 25 or 50 µg/ml 5-Fu induced longer DNA tails and higher tail-DNA content in HCT-8-shB7-H3 vs. HCT-8-NC cells. Percent TDNA, TL, TM and OTM were quantitatively analyzed by CASP software; results are listed in Tables I (corresponding to Fig. 3A) and II (corresponding to Fig. 3B).

Fig. 4A depicts western blot analysis demonstrating the effect of knocking down BRCC3 protein expression with either siRNA1 or siRNA2 (two different siRNA sequences targeting BRCC3); representative immunofluorescence images depicting BRCC3 knockdown are shown in Fig. 4B.

The DNA comet assay shown in Fig. 4C indicates that 5-Fu (25 µg/ml) elicits greater DNA damage in SW480-B7-H3 cells transfected with BRCC3 siRNA1 vs. scramble control siRNA (Fig. 4C). The TDNA%, TL, TM and OTM results are recorded in Table III. Further results indicated that knocking down BRCC3 expression in SW480-B7-H3 cells effectively suppressed colony formation (Fig. 4D). Collectively, our data showed that downregulated BRCC3 can induce DNA damage and reduce colony formation, thereby antagonizing two characteristics of B7-H3. These results suggest that BRCC3 may play a key role in 5-Fu resistance and DNA repair activation caused by B7-H3.