The BRCA1-mutated breast cancer HCC1937 cell line was obtained from the Cell Bank of Shanghai Institute of Cell Biology (Shanghai, China). No wild-type BRCA protein was produced in the HCC1937 cell line. The cells were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere with 5% CO₂.

A plasmid containing a full-length BRCA1 cDNA on a pcDNA3.1 backbone was provided by Dr Genze Shao (Peking University Health Science Center, Beijing, China), for the sub-cloning and generation of the mutated expression constructs. The G1763V and L1786P mutations were generated in this plasmid using QuikChange II XL Site-Directed Mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Primer pairs for the G1763V mutagenesis protocol were as follows: 1763 mutation forward, 5′-AGGACAGAAAGATCTTCAGGGTGCTAGAAATCTG-3′ and reverse, 5′-ACCCTGAAGATCTTTCTGTCCTGGGATTCTCTTG-3′. The primer pairs for the L1786P mutagenesis protocol were as follows: 1786 mutation forward, 5′-GGAATGGATGGTACAGCcGTGTGGTGCTTCTGTGG-3′ and reverse, 5′-CCACAGAAGCACCACACgGCTGTACCATCCATTCC-3′. Large-scale DNA preparations were made using the Qiagen Plasmid Maxi Kit (Qiagen, Inc., Valencia, CA, USA). Each plasmid was sequenced entirely to verify their identity. For gene transfection, HCC1937 cells were grown overnight at 37°C and transfected with plasmids using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.

Total RNA was extracted from transfected HCC1937 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Reverse transcription was performed using a total of 1 µg RNA, oligo (dT) 15 primer, and the ThermoSCRIPT reverse transcription kit (Invitrogen; Thermo Fisher Scientific, Inc.). RT-qPCR was performed using SYBR Green PCR Master Mix (Thermo Fisher Scientific, Inc.) on a ABI7500 Real-Time PCR System (Thermo Fisher Scientific, Inc.). qPCR was performed using the following conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 30 sec and 60°C for 1 min. Primers for BRCA1, poly (ADP-ribose) polymerase 1 (PARP) and GAPDH were as follows: BRCA1_F, 5′-AGGTCCAAAGCGAGCAAGAG-3′ and BRCA1_R, 5′-AGGTGCCTCACACATCTGCC-3′; PARP_F, 5′-CCTAAAGGCTCAGAACGACC-3′ and PARP_R, 5′-AGGAGGGCACCGAACACC-3′; and GAPDH_F, 5′-AGGTCGGAGTCAACGGATTTG-3′ and GAPDH_R, 5′-GTGATGGCATGGACTGTGGT-3′. The results of BRCA1 and PARP were normalized to GAPDH. Data were calculated based on 2^−ΔCq, where ΔCq=Cq (Target)-Cq (Reference). Fold change was calculated using the 2^−ΔΔCq method (12).

Proteins were extracted from cells using radioimmunoprecipitation assay buffer containing complete protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Proteins were separated using 8% SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were then blocked with 5% non-fat milk in TBST [15 mM Tris-HCl (pH 7.4), 0.9% NaCl and 0.05% Tween-20 (pH 7.4)] for 1 h at room temperature., and then incubated with primary antibody overnight at 4°C using rabbit monoclonal anti-human BRCA1 antibody (dilution, 1:1,000; A301-377; Bethyl Laboratories, Montgomery, TX, USA), and goat anti-rabbit secondary antibody (dilution, 1:5,000; ZDR-5306; ZSGB-BIO, Beijing, China). Immunoreactive bands were detected using Super Signal West Femto Chemiluminescent Substrate (Merck KGaA, Darmstadt, Germany). The aforementioned experiments were performed at least three times with consistent results.

The cell proliferation assay was performed using cell counting kit-8 assay. In total, 2×10³ cells were cultured in 96-well culture plates. The cells were resuspended in RPMI-1640 medium containing 10% FBS and cultured for 0, 24, 48 or 72 h at 37°C. The number of viable cells was determined by measuring absorbance at 450 nm using FLUOstar OPTIMA (BMG Labtech, Offenburg, Germany), according to the manufacturer's instructions. Each experiment was performed in triplicate.

The cells were fixed in 4% paraformaldehyde for 30 min at room temperature and lysed with 0.2% Triton-X-100 for 15 min. This was followed by the addition of 5% bovine serum albumin (ZLI-9027, ZSGB-BIO, Beijing, China) to the lysate and incubation for 30 min at 37°C. Rabbit monoclonal anti-human BRCA1 antibody (dilution, 1:1,000; A301-377; Bethyl Laboratories) and monoclonal antibody for histone H2A variant X (γH2AX; dilution, 1:100; 05–636-I; EMD Millipore, Billerica, MA, USA) were added to the mixture followed by incubation at 4°C overnight. Cell lysate with 0.01 mol/l phosphate-buffered saline instead of the primary antibody was used as a negative control. Fluorescence-labeled secondary antibodies (dilution, 1:4,000; ZF-0311, ZF-0316; ZSGB-BIO, Beijing, China) were added to the mixture, which was then incubated in the dark at room temperature for 30 min. The mixture was further incubated with DAPI solution at room temperature for 5 min and then mounted in Tris-buffered glycerol solution. The subcellular localization of the proteins was determined using confocal laser scanning microscopy at a magnification of ×12,50.

A total of 3×10⁵ HCC1937 cells were seeded in 6-wells culture plates in RPMI-1640 medium with 10% FBS. After 12 h, cells were transfected with the aforementioned plasmids using Lipofectamine 2000. Subsequent to transfection, cells were collected at 24, 48 and 72 h and stained with fluorescein isothiocyanate-conjugated Annexin V (BioVision, Inc., Milpitas, CA, USA) at room temperature for 30 min, followed by staining with propidium iodide (PI) 1 min prior to analysis for apoptosis by FACScan (BD Biosciences, San Jose, CA, USA).

Cell cycle stage was determined by flow cytometry using a cell-cycle assay kit (Ab139418, Abcam, Cambridge, UK). Briefly, HCC1937 cells were harvested by centrifugation with 300 × g, 5 min at 4°C, washed with PBS and fixed with cold 75% ethanol at 4°C overnight. The fixed cells were then stained with PI and RNaseA (Ab139418; Abcam, Cambridge, UK), according to the manufacturer's instruction. Following a 30-min incubation in the dark, fluorescence-activated cells were sorted in a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA). The cells were distinguished as being at the G0/G1, G2/M and S phases of the cell cycle based on the fluorescence intensity, and the distribution of the cell-cycle stage was analyzed using ModFit software (BD Biosciences).

Results were expressed as mean ± standard deviation. One-way analysis of variance was used for statistical comparison among groups. Multiple comparison between the groups was performed using Bonferroni method. P<0.05 was considered to indicate a statistically significant difference.

Two novel BRCA1 missense mutations in the BRCT domain were identified from the cohort of Chinese women with familial breast cancer, consisting of G1763V due to p5407 G>T and L1786P due to p5476 T>C. Protein mutation analysis software, consisting of PolyPhen (http://genetics.bwh.harvard.edu/pph/), SIFT (http://sift.jcvi.org/) and Pmut (http://mmb2.pcb.ub.es:8080/PMut/), was used to predict the impact of these two BRCT mutations on BRCA1 protein and their possible pathogenicity. PolyPhen software predicted that G1763V and L1786P both damage the original protein function. SITF and Pmut software predicted that both mutations were deleterious (Table I).

In order to test and verify the function of G1763V and L1786P mutations, mutation plasmids were constructed from pcDNA3.1-BRCA1 (termed wt BRCA1), and the expression of wild and mutant BRCA1 was examined in the BRCA1-deficiency breast cancer HCC1937 cell line. Fig. 1A shows the schematic diagram of the location of the mutants. The sequences of the mutant constructs were verified by sequencing (Fig. 1B). BRCA1 overexpression was observed both at the mRNA level and protein level in these three groups compared with the control group 24 h subsequent to transfection with wt BRCA1 and two mutants (Fig. 1C and D).

Subsequent to successfully constructing the transient transfection cell model, the effect of BRCA1 mutants on breast cancer cell growth and apoptosis was assessed in vitro. Cell proliferation assay using HCC1937 cells showed that the wild type BRCA1 and both of the two mutants significantly reduced the growth of breast cancer cells compared with the control group (P<0.05; Fig. 2A). Additionally, flow cytometric analysis showed that the wild type BRCA1 and both of the mutants significantly increased cell apoptosis compared with the control group (Fig. 2B and C) 72 h subsequent to transfection. These data indicated that G1763V and L1786P have similar tumor suppressor function to wild-type BRCA1 in vitro.

In order to investigate the function of these two mutants following DNA damage, a DNA damage model was created using γ radiation. Phosphorylation of γH2AX is the most sensitive marker that can be used to examine the DNA damage and the subsequent DNA repair. It was found that γH2AX foci increased significantly in the irradiated group when compared with the groups without irradiation (Fig. 3A). After 8-Gy irradiation, the percentages of cells in the S-phase were 42.1, 42.1 and 40.0% in the wild-type BRCA1, G1763V and L1786P cells, respectively, which were increased compared with the control group with 29.2% (control vs. wt BRCA1, control vs. G1763V, control vs. L1786P; P<0.05; Fig. 3B).

In order to test the optimal concentration of Olaparib, HCC1937 cells were cultured in the presence of 0, 20, 40, 80 or 160 µM Olaparib. After 48 h, it was observed that 80 µM Olaparib produced the maximum suppression of PARP expression by 50% (Fig. 4A). Olaparib (80 µM) was then added into the cell cultures when the transfected cells were adherent. Olaparib increased cell apoptosis in HCC1937 (control 0 µM vs. control 80 µM; P<0.05). However, no synergistic effect between the Olaparib and BRCA1 mutation was noted on cell apoptosis (Fig. 4B).