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Nuclear Location and Cell Cycle Regulation of the BRCA2 Protein1 [CANCER RESEARCH 57. 5485-5488. December 15. 1997] Advances in Brief Nuclear Location and Cell Cycle Regulation of the BRCA2 Protein1 David Bertwistle, Sally Swift, Nicola J. Marston, Laura E. Jackson, Susan Crossland, Mark R. Crompton, Christopher J. Marshall, and Alan Ashworth2 Cancer Research Campaign CeñiréforCell ami Molecular Biology [D. B., S. S., N. J. M.. C. J. M.. A. A.I and Section of Gene Function and Regulation ¡A.A.I, Chester Beatt\ Ltiboratories. The Institute of Cancer Rest-arch, London SW3 6JB, Uniteti Kingdom, and Section of Cell Biology and Experimental Palholox\, Huddow Laboratories, The Institute of Cancer Research. Belmoni. Stilton. Surrey SM2 5NG, United Kingdom ¡L.E. J., S. C.. M. R. CJ Abstract observation implicates BRCA2 in a DNA repair process, and this might explain the nature of the tumor suppressor activity of BRCA2. Women carrying a germ-line mutation in the BRCA1 or BRCA2 genes Here we report the generation and characterization of specific have a high risk of developing breast cancer, and loss of the wild-type antibodies against the BRCA2 protein. We have used these antibodies alíelein tumors suggests that these genes function as tumor suppressor genes. The BRCA2 gene encodes a 3418-amino acid protein with no to analyze the cell cycle regulation and subcellular localization of BRCA2. significant sequence similarity to any known protein. To begin to elucidate the cellular role of BRCA2, we have raised antibodies to the BRCA2 Materials and Methods protein and used these to study its subcellular localization and expression. We show that BRCA2 is a nuclear protein expressed in response to cell Antibodies. Peptides were synthesized and conjugated to adjuvants by proliferation and that BRCA2 expression is initiated before DNA synthe- Severn Biotech Ltd. Polyclonal antibodies against peptides corresponding to amino acids 315-330, 1307-1321. 2017-2031, and 3404-3418 of BRCA2 (denoted antisera A. B, C and D. respectively) were raised in rabbits, and a Introduction monoclonal antibody against a peptide corresponding to amino acids 3386- 3400 of BRCA2 was raised in a rat. Peptides were conjugated to PPD Approximately 5% of breast cancers are thought to be due to a (315-330. 1307-1321, 2017-2031) or KLH (3386-3400 and 3404-3418) and hereditary predisposition to the disease (1). Two breast cancer sus injected into rabbits or rats following standard immunization protocols. Rab ceptibility (BRCA) genes have been isolated, and mutations in these bits injected with PPD-conjugated peptides were pretreated with Bacillus genes account for most families with four or more cases of breast Calmette-Giiérin (12). The anti-/3-tubulin antibody, N357. was from Amer- cancer diagnosed before the age of 60 years (1-3). Women who sham. and the anti-TFUH p89 antibody. S-19, was from Santa Cru/, Biotech inherit loss-of-function mutations in either of these genes have nology. A mixture of a mouse monoclonal antibody (PharMingen; 1400IA) and two rabbit polyclonal antibodies (kind gifts of Sibylle Mittnacht; Ref. 13) roughly an 85% chance of developing breast cancer (4). However, were used to detect the retinoblastoma gene product. Rb. mutation of neither gene seems to play a significant role in sporadic Tissue Culture. Cells were cultured in DMEM supplemented with \CI'7i breast cancer ( 1). PCS and 4 mM glutamine. Insulin was included in MCF7 media (at 10 jig/ml). The BRCA2 gene encodes an extremely large protein of 3418 amino Asynchronous MCF7 cells were seeded at 5 x IO5 cells/10-crn dish, cultured acids with a predicted M, of 384,000 (5). The coding sequence has for 24 h in complete media, and then synchronized at G,,-G, by culture in been of little help in defining a biochemical role for BRCA2, because DMEM alone for 24 h. Cells were released back into the cell cycle by it shows no strong sequence similarity to any other known protein. refeeding with DMEM supplemented with 20% FCS. 4 mM glutamine. and 10 /j.g/ml insulin. Alternatively, G,,-G|-synchronized cells were refed with com However, BRCA2 does contain eight internal BRC repeats that are plete media containing 200 JUMmimosine and cultured for 16 h to synchronize well conserved between several species but are of unknown signifi at the G|-S-phase boundary (14). G,-S-phase-synchronized cells were released cance (6, 7). These repeats are all encoded by the extremely large back into the cell cycle by washing and refeeding with complete media. eleventh exon of the BRCA2 gene (6, 7). Interestingly, mutations in Preparation of Cell Extracts. For whole cell protein extracts, MCF7 cells this region of the gene are associated with a higher risk of ovarian were trypsinized. washed in ice-cold PBS. pelleted, and resuspended in RIPA1 cancer relative to breast cancer (8). buffer [50 mM Tris (pH 8.0), 130 mM NaCl, 50 mM NaF, 1 mM sodium Two potential functions have been proposed for the BRCA2 pro orthovanadate, 0.1% SDS, 0.1% deoxycholate. 1% Triton X-100. and tein. A region encoded by exon 3 contains a 45-amino acid portion 1 X complete protease inhibitors (Boehringerl for 10 min on ice. Insoluble with weak similarity to the transcription factor c-jun that can activate debris was removed from extracts by centrilugation at 13,(XX)x g for 10 min. A BCA protein assay kit (Pierce) was used to quantify the protein content of transcription in yeast when fused to the lexA DNA-binding domain cell extracts to enable the standardization of ¡mmunoprecipitations by protein (9). These observations suggest that BRCA2 may have a role in the content. Immunoprecipitations were typically performed using 10 fil of poly regulation of transcription, as has also been suggested for BRCA1 clonal antiserum to precipitate from 500 ^ig of protein extract. After immu- (10). In a separate study, a small region of the mouse Brca2 protein, noprecipitation. proteins were separated by SDS-PAGE on 6% gels and probed which has 95% identity with human BRCA2, was shown to be able to with the anti-BRCA2 rat monoclonal antibody. Cell fractionation was based on bind to radSl. the eukaryotic equivalent of the recA protein that is a protocol described by Lee et al. (15). MCF7 cells were trypsinized, washed in ice-cold PBS, pelleted, and resuspended in hypotonie buffer [10 mM Hepes- involved in DNA repair and recombination (11). This significant KOH (pH 6.4). 10 mM NaCl. 1.5 mM MgCK, and 1 X complete protease inhibitors (Boehringer) for 10 min on ice to swell. Cells were inspected under Received 9/26/97; accepted 10/29/97. a microscope to ensure they were swollen, and then they were lysed with 25 The costs of publication of this article were defrayed in part by the payment of page strokes in a 0.1-ml Wheaton Dounce homogenizer. Homogenized cells were charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. inspected under a microscope to ensure lysis and then centrifuged immediately ' Supported by the Cancer Research Campaign. at 400 x %for 10 min at 4°C.The supernatant was transferred to another tube : To whom requests for reprints should be addressed, at CRC Centre for Cell and Molecular Biology, Chester Beatty Laboratories. The Institute of Cancer Research. Fulham Road. London SW3 6JB, United Kingdom. Phone: 0171-352-8133; Fax: 0171- ' The abbreviations used are: RIPA, radio radioimmunoprecipitation assay; IP. immu- 352-3299: E-mail: [email protected]. noprecipitation. 5485 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1997 American Association for Cancer Research. THE HKCA2 GENE PRODUCT and centrifugal again, this time at 13,000 X ¡>forIO min. This supernatant was adjusted to 1 X RIPA buffer and taken as the cytoplasm/membrane fraction. The nuclear pellet from the immediate postlysis centrifugaron was resus- pended in RIPA for IO min on ice and then centrifuged for IO min at 13.(XX) x g. The supernatant was taken as the nuclear fraction. Immunopre- cipitations of fractions were standardized by cell number. Fluorescence-activated Cell-sorting Analysis. Cells were harvested by trypsinising. then washed in PBS. fixed in l()c/r ethanol. washed in PBS again, and then resuspended in PBS containing 100 /¿g/mlRNAse A and 40 /¿g/ml propidium iodide for 30 min at 37°C.Propidium iodide-stained cells were —¿BRCA2 analy/.ed using a FACScan (Becton Dickinson I. A minimum of H).(XX)cells were analyzed per time point. Results -TFIIH p89 Identification of the BRCA2 Protein. To study the expression of the BRCA2 protein, we used antibodies raised in rabbits to four different peptides and a rat monoclonal antibody raised against a fifth —¿ÃŸ-tubulin peptide of human BRCA2 (Fig. \A). To ensure specificity for BRCA2. combinations of these antibodies were used in an IP/Western protocol. Immunoprecipitates of MCF7 breast cancer cells generated Fig. 2. BRCA2 is a nuclear prolein. Immunoprecipitates from MCF7 total cell lysates or troni the nuclear or the cytoplasmic and membrane fractions of MCF7 cells with anli-BRC'A2 serum B were separated by SDS-PAGE. Western hlotied. and probed with the rat monoclonal antibody to BRCA2. As controls for the fraelionation procedure, total cell lysate and the fractions were separated by SDS-PAGE. Western blotted, and probed with antibodies raised against TFIIH p89 or ß-lubulin as standards for nuclear and cyloplasmie proteins, respectively. using the rabbit antibodies were separated by SDS-PAGE, Western blotted, and analyzed with the rat monoclonal antibody. A single large •¿Â£On n m nnrnU protein of the same size was detected in immunoprecipitates produced ULUT U ULJU with all four rabbit antipeptide antibodies (Fig.
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