Mutations in BRCA1 and BRCA2 account for the majority of familial breast cancers. Cells with mutated BRCA1 or BRCA2 are hypersensitive to ionizing radiation (IR) and exhibit defective DNA repair. Both BRCA1 and BRCA2 have been reported to bind Rad51, a protein essential for homologous recombination and the recombinational repair of DNA double-strand breaks. In normal cells, a redistribution of Rad51 protein, manifested as formation of Rad51 nuclear foci, is seen upon IR treatment. Here we demonstrate that IR-induced Rad51 foci formation is aberrant in BRCA2- but not BRCA1-deficient tumor cells. In Capan-1 cells, which do not express functional BRCA2, there was little Rad51 foci formation in response to a wide range of radiation dosages. Moreover, forced expression of a fusion protein containing green fluorescent protein and the first Rad51-binding BRC repeat of BRCA2 in cells with wild-type BRCA2 rendered them hypersensitive to IR and cisplatin and compromised IR-induced Rad51 foci formation. In HCC1937 cells, which harbor mutation of BRCA1, IR-induced Rad51 foci were readily detected. This study suggests a requirement of BRCA2 protein for the IR-induced assembly of Rad51 complex in vivo.

Germ-line mutation of BRCA1 and BRCA2accounts for the majority of the familial breast cancers (Ref. 1 and references therein). Most of the tumors from the predisposed individuals show loss of the wild-type allele, consistent with Knudson’s “two hits” hypothesis of the recessive tumor suppressor genes (2). Although the protein products encoded by these two genes share little homology in their amino acid sequences, both of the two proteins have been implicated in DNA repair. Mouse cells harboring mutations in both alleles of BRCA1 are hypersensitive to IR4 and exhibit defective transcription-coupled DNA repair (3, 4). Cells with the wild-type BRCA2 gene become sensitive to IR when the expression of BRCA2 is decreased by treatment with antisense BRCA2 oligonucleotides (5). MEFs carrying insertional mutations that delete the BRCA2 COOH terminus are hypersensitive to IR (6, 7, 8, 9). BRCA2-deficient MEFs maintain the G1-S and G2-M checkpoints after exposure to IR, suggesting that defective DNA repair is the main cause of radiosensitivity of these cells (8). In agreement with this suggestion, Connor et al.(6) demonstrated defective DNA DSB repair in BRCA2-deficient MEFs. In addition to mouse cells, the human pancreatic adenocarcinoma Capan-1 cells, which express a COOH terminus-truncated BRCA2 protein of Mr 224,000 (5, 10, 11), are hypersensitive to DNA-damaging agents mitoxantrone, amsacrine, etoposide, and MMS (5, 10). Evidently, Capan-1 cells exhibit a slower rate of DSB repair than cells with wild-type BRCA2 (5). Introduction of wild-type BRCA2 into Capan-1 cells corrected their sensitivity to MMS (10), indicating that the hypersensitivity of Capan-1 cells to MMS is due to the BRCA2 mutation. Taken together, these studies demonstrate a crucial role of BRCA1 and BRCA2 in DNA DSB repair. However, the mechanisms underlying their functions in DNA repair are not clear.

In mammalian cells, DSB are repaired by end-joining and homologous recombination (12). Rad51 is a pivotal player of a large protein complex that functions in homologous recombination and recombinational repair of DSB (13). Biochemical studies have demonstrated that Rad51 is a recombinase that oligomerizes on DNA and promotes pairing and strand exchange between homologous DNA (Ref. 13 and references therein). In immunostaining experiments, the Rad51 protein is found in discrete nuclear dots, or foci, upon induction of DSBs (14). Because Rad51 foci colocalize with single-strand DNA, which is formed by the processing of double-strand DNA at the DSB, the Rad51 foci likely represent functional, multimeric forms of Rad51 that promote DNA repair (15). Recent studies begin to reveal the biochemical composition of Rad51 foci and protein factors required for formation of these foci. Studies using yeast mutants have demonstrated that multiple DNA recombination proteins including Rad52, Rad55, and Rad57 are not only present in the Rad51 foci but also required for the appearance of Rad51 foci (16). In Chinese hamster ovary cells, Xrcc3 is necessary for normal DNA DSB repair and Rad51 foci formation (17). These studies also demonstrate a strong correlation between defective DSB repair and defective Rad51 foci formation, suggesting that IR-induced Rad51 foci is a crucial cellular response to DNA damage.

Recently, BRCA1 and BRCA2 have been shown to bind Rad51 and colocalize with Rad51 foci after IR (18, 19). The regions of BRCA2 that interact with Rad51 have been mapped to the eight BRC repeats (10, 20). The eight BRC repeats in BRCA2 appear to be redundant for Rad51 binding, because any one of the first four BRC repeats could bind to Rad51 efficiently (10, 20). Both the BRC repeat and the COOH terminus of BRCA2 are essential for normal sensitivity to DNA-damaging agents (8, 10), suggesting that BRCA2-Rad51 interaction is functionally significant. Here, we investigated whether BRCA2 is involved in IR-induced Rad51 foci formation.

Cell Culture.

Capan-1, BxPC-3, MCF10A, and COS-7 cell lines were obtained from the American Type Culture Collection. HCC1937 was established from tumor samples of a patient carrying germ-line mutation of BRCA1 (21). Capan-1 and HCC1937 cells were cultured in RPMI 1640 containing 15% fetal bovine serum and 1 mm glutamine. MCF10A cells were grown in DMEM F-12 containing 5% horse serum, 1% penicillin streptomycin, 0.14% hydrocortisone, 1% insulin, 0.1% choleratoxin, 0.2% epidermal growth factor, and 1 mm glutamine. COS-7 cells were maintained in DMEM containing 10% fetal bovine serum.

Immunostaining.

Subconfluent cultures of Capan-1, BxPC-3, MCF10A, and HCC1937 cells grown in normal medium were placed in 0.1% serum. One day later, the cultures were irradiated with 10 Gy of γ-rays using a 137Cs gamma cell. The cells were immediately returned to the tissue culture incubator and were fixed with 4% paraformaldehyde at various time points after irradiation. After permeabilization with 0.2% Triton X-100 in TBST [50 mm Tris (pH 7.4), 150 mm NaCl, and 0.05% Tween 20] and blocking with 4% milk in TBST, the cells were incubated with a monoclonal antibody against human Rad51 or Rad50 and then with either FITC or Texas Red-conjugated goat anti-mouse IgG (Jackson Immunochemicals). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole. Rad51 and Rad50 foci were examined under a Zeiss AXIOPHOT fluorescence microscope.

Immunoblotting and Immunoprecipitation.

Cells in monolayer cultures were collected by scraping with a rubber policeman. The cells were lysed in ice-cold lysis buffer [50 mm Tris (pH 7.4), 120 mm NaCl, 1 mm EDTA, 0.5% NP40, and 50 mm NaF] plus protease inhibitors. The extracts were clarified by centrifugation at 16,000 × g for 10 min at 4°C. Protein concentrations were quantified by the Bradford assay (22). One hundred μg of proteins were separated in SDS-PAGE and then transferred to nitrocellulose membrane. The blots were blocked with 4% milk in TBST and incubated with primary antibody. After three washes with TBST, the blots were incubated with secondary antibodies conjugated with alkaline phosphatase. Antigen-antibody complexes were detected by color reactions with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. Immunoprecipitation of GFP or BRC-GFP were performed using a rabbit polyclonal anti-GFP antibody (Clontech) and protein G-agarose as described by Harlow and Lane (23).

Expression of BRC-GFP.

Reverse transcription-PCR was performed to obtain a DNA fragment of BRCA2 that encodes the first BRC repeat (amino acids 927-1172). The PCR product was cloned into pEGFP-N1 (Clontech) to allow in-frame fusion of GFP to the BRC COOH terminus. COS-7 cells were transfected with either pEGFP-N1 or pBRC-EGFP by FuGene (Boehringer Mannheim), following the manufacturer’s instructions. Two days later, the transfected cells were harvested for analysis of BRC-GFP protein expression.

Capan-1 cells lack one copy of the BRCA2 gene and carry a 6174delT mutation in the other allele (5, 11). HCC1937 is a human breast carcinoma cell line established from a germ-line BRCA1 mutation carrier. The BRCA1 gene in HCC1937 cells is mutated after codon 1755 (21). As a result, a truncated BRCA1 protein is expressed in these cells (19). We used Capan-1 and HCC1937 cells to test whether IR-induced Rad51 foci requires wild-type BRCA1 and/or BRCA2. The human pancreatic adenocarcinoma BxPC-3 cells and the breast epithelial MCF10A cells served as controls in these experiments. Two h after treatment with 10 Gy of IR, Rad51 foci were readily detected in BxPC-3, MCF10A, and HCC1937 cells but not in Capan-1 cells (Fig. 1,a). Specifically, 38 and 68% of BxPC-3 cells contained detectable Rad51 foci at 2 and 12 h after IR treatment, respectively (Fig. 1,b). The percentages of HCC1937 and MCF10A cells containing Rad51 foci 2 and 12 h after IR treatment were similar to BxPC-3 cells (Fig. 1,b). In contrast, only 5 and 6% of the irradiated Capan-1 cells displayed detectable Rad51 foci at 2 and 12 h after IR, respectively (Fig. 1,b). In another experiment, Capan-1 and BxPC-3 cells were treated with 0.5, 2, and 10 Gy of IR. The cells were stained for Rad51 foci 2 h after IR treatment. Rad51 foci were readily detected in BxPC-3 cells after all three dosages of IR (Fig. 1,c). The number of BxPC-3 cells containing detectable Rad51 foci increased with the IR dosage (Fig. 1,c). HCC1937 cells also exhibited an IR dosage-dependent Rad51 foci formation (data not shown). In contrast, Capan-1 cells showed little increase in Rad51 foci formation 2 h after treatment with the three dosages of IR (Fig. 1 c). These results demonstrate that IR-induced Rad51 foci formation occurs in the BRCA1 mutant HCC1937 cells but is defective in the BRCA2 mutant Capan-1 cells.

In addition to Rad51 foci, IR also induces the DSB repair protein complex of Rad50, Mre11, and nibrin/p95 to form nuclear foci (24). Biochemical studies have demonstrated that this complex possesses exonuclease activity (25, 26). Significantly, Rad50 foci have been shown not to colocalize with Rad51 foci (27). To address whether the effect of BRCA2 mutation on Rad51 foci formation is selective, we examined Rad50 foci in Capan-1 cells upon IR. Capan-1 and the control BxPC-3 cells were treated with 10 Gy of IR. Two h later, the cells were stained with an antibody against Rad50 (Fig. 2,a). Rad50 foci were formed in Capan-1 cells as proficiently as in BxPC-3 cells (Fig. 2,b). Approximately 50% of both Capan-1 and BxPC-3 cells displayed Rad50 foci (Fig. 2 b). This result suggests that the BRCA2 mutation in Capan-1 cells does not affect IR-induced Rad50 foci formation.

To ascertain whether the reduced Rad51 foci formation in Capan-1 cells could be attributed to the level of Rad51 protein in these cells, protein extracts from BxPC-3, Capan-1, HCC1937, and MCF10A cells were subjected to immunoblotting with the anti-Rad51 antibody. As a protein loading control, the blot was also probed with an antibody against p84, a nuclear GTP-binding protein expressed at constant levels in many cell types (28). BxPC-3, Capan-1, and HCC1937 cells expressed similar levels of Rad51 protein (Fig. 3). There was little change in Rad51 protein level after IR treatment in these cells (Fig. 3). MCF10A cells expressed less Rad51 protein (Fig. 3) but were nevertheless proficient in Rad51 foci formation (Fig. 1). Taken together, these results indicate that defective IR-induced Rad51 foci formation in Capan-1 cells is not due to Rad51 protein level.

BRCA2 protein contains eight evolutionarily conserved BRC repeats. A single BRC motif of BRCA2 is sufficient for binding to Rad51 (10, 20). To address whether the BRC motif of BRCA2 is involved in Rad51 foci formation upon IR, a fusion protein composed of GFP and the first BRC repeat of BRCA2 was expressed in COS-7 cells by transient transfection. COS-7 cells were used here because of their high transfection efficiency and the high degree of conservation among mammalian Rad51 (13) and among mammalian BRCA2 BRC motif. As a control, COS-7 cells were also transfected with wild-type GFP. Immunoprecipitation followed by immunoblotting with anti-GFP antibodies confirms the expression of the Mr 55,000 BRC-GFP fusion protein (Fig. 4,a). The cells expressing GFP or BRC-GFP were examined for the ability to form Rad51 foci 2 h after 10 Gy of IR. Rad51 foci were readily formed in GFP-positive cells and to a much lesser extent in BRC-GFP-positive cells (Fig. 4,b). Approximately 50% of GFP-positive cells contained detectable Rad51 foci 2 h after IR treatment (Fig. 4,c). In contrast, only 15% of BRC-GFP-positive cells contained detectable Rad51 foci 2 h after IR treatment (Fig. 4 c). Thus, expression of a single BRC motif of BRCA2 in COS-7 cells could reduce Rad51 foci formation upon IR. Similar to COS-7 cells, expression of BRC-GFP in the human 293T cells also reduced Rad51 foci formation upon IR (data not shown). These results suggest that overexpression of the BRC repeat of BRCA2 may have a dominant-negative effect, because it compromised IR-induced Rad51 foci formation in wild-type cells. Although the mechanism is not clear, these experiments suggest that the BRCA2 BRC motif is involved in nuclear redistribution of Rad51 protein after IR treatment.

The inability to efficiently form Rad51 foci in cells overexpressing BRC-GFP prompted us to investigate whether these cells are hypersensitive to DNA-damaging agents. Transfected COS-7 cells expressing GFP or BRC-GFP were treated with 10 Gy of IR and 10 mm cisplatin. One day later, the cultures were fixed, and dying cells with pyknotic nuclei were scored. Ten Gy of IR treatment resulted in cell death in 13% of GFP-expressing cells and 55% of BRC-GFP-expressing cells (Fig. 4,d). In addition to hypersensitivity to IR, the cells expressing BRC-GFP were also significantly sensitive to cisplatin. Treatment with 10 mm cisplatin resulted in cell death in 16% of GFP-expressing cells and 62% of BRC-GFP-expressing cells (Fig. 4 d). This observation suggests that overexpression of a BRCA2 BRC repeat may not only interfere with Rad51 foci formation but also render cells hypersensitive to DNA damage.

HCC1937 cells with mutant BRCA1 were proficient in Rad51 foci formation. It is possible that the truncated BRCA1 protein expressed in these cells acts to mediate Rad51 foci formation (19). Alternatively, BRCA1 may affect other but not Rad51-mediated repair pathways. The reduced Rad51 foci response to IR in Capan-1 cells and the effects of overexpression of the BRCA2 BRC motif on Rad51 foci formation collectively suggest the importance of wild-type BRCA2 for this process. Because a total of six Rad51-binding BRC repeats are present in the BRCA2 (20), we speculate that BRCA2 may facilitate the oligomerization of Rad51 and/or enhance its interaction with other repair proteins, such as Xrcc3 and Rad54 in response to IR. Recent studies demonstrate the enhanced interaction between Rad51 and Rad54 in response to IR. Interestingly, Rad54 is also required for Rad51 foci formation in mammalian cells (29). Capan-1 cells express a truncated BRCA2 protein that contains the BRC repeats (10), consistent with the coimmunoprecipitation of Rad51 and BRCA2 in Capan-1 cells (19). Nevertheless, Capan-1 cells do not form Rad51 foci efficiently, indicating that the COOH terminus of the BRCA2 is also crucial for Rad51 foci formation. The present study suggests a role for the BRCA2 COOH terminus in cellular processes contributing to DSB repair. Additional studies are required to understand how BRCA2 protein is involved in the IR-induced assembly of the Rad51 complex in vivo.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

      
1

This work was supported by Breast Cancer Specialized Programs of Research Excellence Grant 5P50CA58183-07, Grant ATP3659-034 from Texas Advanced Research/Advanced Technology Program, and Grant 1R01NS378381-01 from the NIH (to E. Y-H. P. L.). G. C. is a recipient of a NIH postdoctoral fellowship.

                  
4

The abbreviations used are: IR, ionizing radiation; DSB, double-strand break; MEF, mouse embryonic fibroblast; MMS, methanesulfonate; GFP, green fluorescent protein.

Fig. 1.

Rad51 foci formation in BxPC-3, Capan-1, HCC1937, and MCF10A cells upon IR. a, Rad51 foci in cells without IR (−IR) and 2 h after IR (+IR) treatment detected by immunostaining with a monoclonal anti-Rad51 antibody. b, histogram depicting the percentages of nuclei containing detectable Rad51 foci without IR treatment and 2 and 12 h after IR treatment. c, IR dosage dependence of Rad51 foci in Capan-1 and BxPC-3 cells. The cells were treated with 0, 0.5, 2, and 10 Gy of IR and were stained for Rad51 foci 2 h later. At least 200 cells were scored for each data point in b and c.

Fig. 1.

Rad51 foci formation in BxPC-3, Capan-1, HCC1937, and MCF10A cells upon IR. a, Rad51 foci in cells without IR (−IR) and 2 h after IR (+IR) treatment detected by immunostaining with a monoclonal anti-Rad51 antibody. b, histogram depicting the percentages of nuclei containing detectable Rad51 foci without IR treatment and 2 and 12 h after IR treatment. c, IR dosage dependence of Rad51 foci in Capan-1 and BxPC-3 cells. The cells were treated with 0, 0.5, 2, and 10 Gy of IR and were stained for Rad51 foci 2 h later. At least 200 cells were scored for each data point in b and c.

Close modal
Fig. 2.

Rad50 foci formation in Capan-1 and BxPC-3 cells without IR treatment and 2 h after treatment with 10 Gy of IR. a, immunostaining of Rad50 foci. b, histogram shows the percentages of cells with Rad50 foci. At least 200 cells were scored for each data point.

Fig. 2.

Rad50 foci formation in Capan-1 and BxPC-3 cells without IR treatment and 2 h after treatment with 10 Gy of IR. a, immunostaining of Rad50 foci. b, histogram shows the percentages of cells with Rad50 foci. At least 200 cells were scored for each data point.

Close modal
Fig. 3.

Rad51 protein expression in cells without IR treatment and 2 h after 10 Gy of IR treatment. Extracts of the cells were subjected to immunoblotting with antibodies against Rad51 and p84.

Fig. 3.

Rad51 protein expression in cells without IR treatment and 2 h after 10 Gy of IR treatment. Extracts of the cells were subjected to immunoblotting with antibodies against Rad51 and p84.

Close modal
Fig. 4.

Expression of BRC-GFP compromises IR-induced Rad51 foci formation. a, expression of BRC-GFP and GFP in COS-7 cells by transient transfection. Extracts from mock-transfected cells and cells transfected with the indicated plasmids were subject to immunoprecipitation with a rabbit polyclonal anti-GFP antibody. The immunoprecipitates were then analyzed by immunoblotting with a mouse monoclonal anti-GFP antibody. Arrow and arrowhead, apparently full-length BRC-GFP and GFP proteins, respectively. b, immunostaining of COS-7 cells transfected with either GFP or BRC-GFP 2 h after 10 Gy of IR treatment with anti-Rad51 antibody. Green-colored cells represent cells expressing GFP or BRC-GFP. Rad51 protein was stained red. Arrows, GFP-positive cells that contained Rad51 nuclear foci. Arrowheads, GFP or BRC-GFP-positive cells that lacked Rad51 nuclear foci. c, histogram showing the percentages of nuclei with Rad51 foci in GFP or BRC-GFP-positive cells without IR treatment and 2 h after 10 Gy of IR treatment. d, histogram showing the percentages of dying cells among cells expressing GFP and BRC-GFP with and without IR or cisplatin treament. COS-7 cells were treated with 10 Gy of IR or 10 mm cisplatin 2 days after transfection. The cultures were stained with 4′,6-diamidino-2-phenylindole 1 day later. Dying cells were judged by the appearance of pyknotic nuclei. At least 200 cells were scored for each data point in c and d.

Fig. 4.

Expression of BRC-GFP compromises IR-induced Rad51 foci formation. a, expression of BRC-GFP and GFP in COS-7 cells by transient transfection. Extracts from mock-transfected cells and cells transfected with the indicated plasmids were subject to immunoprecipitation with a rabbit polyclonal anti-GFP antibody. The immunoprecipitates were then analyzed by immunoblotting with a mouse monoclonal anti-GFP antibody. Arrow and arrowhead, apparently full-length BRC-GFP and GFP proteins, respectively. b, immunostaining of COS-7 cells transfected with either GFP or BRC-GFP 2 h after 10 Gy of IR treatment with anti-Rad51 antibody. Green-colored cells represent cells expressing GFP or BRC-GFP. Rad51 protein was stained red. Arrows, GFP-positive cells that contained Rad51 nuclear foci. Arrowheads, GFP or BRC-GFP-positive cells that lacked Rad51 nuclear foci. c, histogram showing the percentages of nuclei with Rad51 foci in GFP or BRC-GFP-positive cells without IR treatment and 2 h after 10 Gy of IR treatment. d, histogram showing the percentages of dying cells among cells expressing GFP and BRC-GFP with and without IR or cisplatin treament. COS-7 cells were treated with 10 Gy of IR or 10 mm cisplatin 2 days after transfection. The cultures were stained with 4′,6-diamidino-2-phenylindole 1 day later. Dying cells were judged by the appearance of pyknotic nuclei. At least 200 cells were scored for each data point in c and d.

Close modal

We thank Patrick Sung for critical reading of the manuscript. We are grateful to Wen-Hwa Lee and Shang Li for stimulating discussions.

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