Nuclear Location and Cell Cycle Regulation of the BRCA2 Protein1

[CANCER RESEARCH 57. 5485-5488. December 15. 1997] Advances in Brief

David Bertwistle, Sally Swift, Nicola J. Marston, Laura E. Jackson, Susan Crossland, Mark R. Crompton, Christopher J. Marshall, and Alan Ashworth2

Cancer Research Campaign CeÃ±irÃ©forCell ami Molecular Biology [D. B., S. S., N. J. M.. C. J. M.. A. A.I and Section of Gene Function and Regulation Â¡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 Â¡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 alÃelein 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-GiiÃ©rin (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 Â¡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 4Â°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 Â¡>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 resuspended 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 /Â¿g/mlRNAse A and 40 /Â¿g/ml propidium iodide for 30 min at 37Â°C.Propidium iodide-stained cells were â€”¿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 â€”¿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 ÃŸ-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€¢¿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. IÃŸ).This protein was Ã¯A T not detected when preimmune serum was used for immunoprecipita- B CÂ«fnuRmAb D tion. Furthermore, the protein was not detected in CAPAN 1 cells that carry the 6174delT BRCA2 mutation common in the Ashkenazi 100 amino acids Jewish population and that have lost the wild-type BRCA2 alÃele(16). This mutation would result in loss of the epitope that the antibody B used to probe the Western blot was raised against. These results indicate that the protein detected by all five of the antibodies in MCF7 <<0- f- < f- < f*. Q_< LL Q_ Ãœ- ih Â£ih0 cells is the BRCA2 gene product. CELL MCF7 cells were fractionated into nuclear and cytoplasm/membrane O1234567f*~Q. < ffl Q. CD o. O O o. O 8 9 10 11 12 fractions. Western blots of the fractions were probed with antibodies raised against TFIIH p89 and ÃŸ-tubulinas controls for nuclear and cytoplasmic proteins, respectively (Fig. 2). The fractions were ana -BRCA2 lyzed for BRCA2 by the IP/Western procedure described above, using anti-BRCA2 serum B for immunoprecipitation. This revealed that BRCA2 was localized predominantly to the nuclear fraction (Fig. 2). 220 kDa- Expression of the BRCA2 Protein Is Regulated by the Cell Cycle. As a first step in analyzing the regulation of the BRCA2 protein, we have examined its expression through the cell cycle. MCF7 cells synchronized in G,, by serum starvation were released back into the cell cycle by refeeding with complete media and ana lyzed at various times after serum stimulation. BRCA2 protein levels were assayed using the IP/Western protocol described above, immu- noprecipitating with anti-BRCA2 serum B. The cell cycle profile of 97 kDa - the cells was monitored by How cytometry of propidium iodide- stained cells and by probing Western blots of cell extracts for the retinoblastoma gene product, Rb. Control cells growing in complete Fig. I. Identification of the BRCA2 prolein. A. diagram of the BRCA2 protein media expressed an easily detectable level of BRCA2 protein (Fig. indicaling Ihe positions of the peplides the four rabbit polyclonal antisera (A-D) and one rat monoclonal antibody (mAbl were raised against, fi. immunoprecipitates of MCF7 3A, Lane 1). However, after serum starvation for 24 h, the protein was (Lanes 2. 3. 5, 6. 8. 9. II, and 12). and CAPAN1 cells (Lanes I, 4. 7. and 10) separated at much lower levels (Fig. 3/4, Lane 4). Therefore, expression of by SDS-PAGE, Western blotted, and probed with the rat monoclonal antibody to the BRCA2 seems to be associated with cell proliferation. At approxi BRCA2 protein. Immunoprecipitations were performed using rabbit polyclonal antisera A (Lanes 1 and .ÃŽ).B (Lanes 4 and 6), C (Lanes 7 and 9), D (Lanes 10 and 12). or their mately 12 h after serum stimulation, BRCA2 protein levels start to corresponding preimmune sera (Lanes 2, 5. 8. and //). increase (Fig. 3A, Lane 7). At this time, propidium iodide staining 5486

mimosine-blocked cells confirmed that they have not initiated DNA synthesis (Fig. 4ÃŸ).Therefore, BRCA2 is induced at the G,-S-phase U. boundary before DNA synthesis. Ã¼2Iflu Discussion after serum stimulation The BRCA1 and BRCA2 genes both encode very large novel pro MCF7i-of GO synchronized "Z.1 teins about which little is known. Careful characterization of these jCfjCjccMuno^-cocMi- t- JC JC JC jC jC proteins will be essential to define their biochemical roles and explain â€¢¿growth factor -..--.tocoAsynchronousCo/GÃ¬ receptor ( 17-21 ). To minimize the chances of spurious results due to cross-reactivity with other proteins, we have used an IP/Western 12Hours5 6 7 8 9 10 11 procedure to identify the BRCA2 protein. In this procedure, a protein must be bound by two antibodies recognizing distinct epitopes to be B detected. This minimizes the chances of cross-reactivity with unre lated proteins. stimulationi_i_after serum All four combinations of antibodies we have used detect a single large protein of the same size. The size of the protein detected is v_ .C .C -C _C .C .C

II89II II II II II II M II

61 90 92 82 68 34 26 53 67

S21G2/M 5 5 3 11 24 46 45 13 17 (DjOÂ£ 15Asynchronous>r-P-Rb{5 515Distribution 4 7 7 19 29 33 .1

CHype EE 3 2* -? ?r^rC stimulation,_Hours after serum Â¿^ ? JE m O co i_ |_JC JCJCJCJCJC JL1GO/GIsG2/IV-< co JL JC -C .C CN UDO^tOOCN OCOII â€¢¿*!â€¢00â€¢¿>-T- CM CM CM II-!*â€¢â€¢â€¢â€¢II II II II II II II --2345C/9 BRCA2>e Â£ HypN.o-P-Rb-4

Fig. 3. Cell cycle regulation of BRCA2. A, immunoprecipitates from unsynchronized B MCF7 cells (Lanes 1 and 2). CAPAN I cells (Lane 3), or from MCF7 cells serum- uoQO)0EEEo^co30663 stimulated for 0. 4, 8. 12, 15, 20, 24, 28, or 32 h (Lanes 4-12, respectively) after first .CDE.kpCDco.ra being serum-starved for 24 h were generated using anti-BRCA2 serum B (Lanes I, and 3-12) or preimmune serum (Lane 2), separated by SDS-PAGE. Western blotted, and probed with the rat monoclonal antibody lo BRCA2. B and C. cell cycle profile of Go-synchronized MCF7 cells at various times after serum stimulation. B. flow cylometry of propidium iodide-stained cells. C. Western blot of cell extracts probed with antibodies roo raised against Rb. ~30Â¡2C 0i I0 T3 r^c CU indicated that cells were starting to progress into S phase (Fig. 3fi). In Â£Â¿III44 agreement with this, a shift from the GirG,-specific hypophosphory- lated form of Rb to the hyperphosphorylated form that exists during 899031 cell cycle progression (13) occurred between 8 and 12 h after serum stimulation (Fig. 3C). Thus, induction of BRCA2 seemed to coincide 4524 with the entry of cells into S phase. 64â€” To determine whether BRCA2 induction is dependent on DNA Distribution (%) synthesis, we have used the replication inhibitor mimosine. Mimosine can synchronize cells released from serum starvation at the G,-S- Fig. 4. BRCA2 is induced before DNA replication. A. immunoprecipitates from phase boundary (14). MCF7 cells synchronized at the G,-S-phase asynchronous MCF7 cells (Lanes I and 2). MCF7 cells serum-starved for 24 h (Lane 3). MCF7 cells starved then refed with media including mimosine for 16 h (iMiit1 4). and boundary in this way (Fig. 4/4, Lane 4) have similar levels of BRCA2 MCF7 cells starved, refed with media including mimosine. and then released into protein to asynchronous MCF7 cells (Fig. 4/4, Lane 2) and to MCF7 complete media for 3 h (Lane 51. Immunoprecipitates were generated with anti-BRCA2 cells 3 h after release from the G,-S-phase boundary into S phase (Fig. serum B (Lanes 2-5) or preimmune serum (Lane /I. separated by SDS-PAGK. Western blotted, and probed with the rat monoclonal antibody to BRCA2. H, cell cycle profile of 4/4, Lane 5) but much higher levels of BRCA2 protein than serum- the asynchronous and treated MCF7 cells from flow cytometry of propidium iodide- starved MCF7 cells (Fig. 4/4, Lane 3). Propidium iodide staining of stained cells. 5487

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1997 American Association for Cancer Research. THE BKCA2 GENE PRODUCT difficult to measure accurately, but our estimates are consistent with strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science (Washington DCI, 266: 66-71, 1994. the size predicted to be encoded by the BRCA2 gene (5). Furthermore, 3. Wooster. R.. Bignell. G.. Lancaster, J., Swift, S.. Seal. S.. Mangion. J.. Collins, N., this protein comigrates with flag-tagged, full-length BRCA2 ex Gregory. S.. Gumbs. C.. Micklem. G.. Barfoot, R.. Hamoudi. R.. Palei. S.. Rice, C, pressed in COS cells from an expression plasmid (data not shown). Biggs. P., Hashim. Y., Smith, A.. Connor. F.. Arason. A., Gudmundsson. J., Ficenec. D.. Kelsell, D., Ford, D., Tonin, P., Bishop, T., Spurr, N. K., Ponder, B. A. J., Eeles, Therefore, in MCF7 cells, at least, BRCA2 seems to exist primarily as R., Peto, J.. Devilee, P.. Cornelisse, C., Lynch. H.. 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S.. Janecki. T.. Jiang. P.. Kehrer. R.. Leblanc, J. F.. Mitchell. J. T.. involved in DNA repair and recombination (II)4 and Brca2-/- blasto- McArthur-Momson. J., Nguyen. K., Peng, Y.. Samson, C.. Schroeder. M.. Snyder. S. C., Steele. L.. Stringfellow. M.. Stroup. C.. Swedlund, B.. Swensen. J., Teng. D., cysts are hypersensitive to ionizing radiation (11). Furthermore, fibro- Thomas. A.. Tran, T., Tran. T.. Tranchant. M.. Weaver-Feldhaus. J.. Wong. A. K. C.. blasts from Brca2-7- mouse embryos are deficient in DNA repair.5 If Shizuya. H.. Eyfjord, J. E.. Cannon-Albright. L., Labrie. F.. Skolnick. M. H.. Weber. B.. Kamb. A., and Goldgar. D. E. The complÃ¨te BRCA2 gÃ¨ne and mutations in Brca2 participates in a DNA repair process, then it would need to be chromosome 13q-linked kindreds. Nat. Genet., 12: 333-337, 1996. present in the nucleus. The ability of part of human BRCA2 to activate 6. Bork. P.. Blomberg. N., and Nilges. M. Internal repeats in the BRCA2 protein transcription in yeast when fused to the lexA DNA-binding domain also sequence. Nat. Genet., 13: 22-23, 1996. 7. Bignell. G.. Micklem, G.. Stratton, M. R., Ashworth. A., and Wooster, R. The BRC suggests a role for BRCA2 in the nucleus (9). Therefore, our observation repeats are conserved in mammalian BRCA2 proteins. Hum. Mol. Genet., 6: 53-58, that BRCA2 is localized to the nucleus of MCF7 cells is consistent with 1997. current data concerning the cellular roles of BRCA2. 8. Gayther. S. A., Mangion. J.. Russell, P.. Seal, S.. Barfoot. R.. Ponder. B. A., Stratton, M. R.. and Easton, D. Variation of risks of breast and ovarian cancer Previous work has demonstrated cell cycle regulation of the BRCA2 associated with different germline mutations of the BRCA2 gene. Nat. Genet., /5: mRNA (22). Because transcription and translation of a gene are not 103-105, 1997. 9. Milner. J.. Ponder. B.. Hughes-Davies, L.. Seltmann. M.. and Kouzarides. T. Tran necessarily coregulated, we have asked whether expression of the scriptional activation functions in BRCA2. Nature (Lond.), 386: 772-773. 1997. BRCA2 protein is regulated through the cell cycle. We find that 10. Chapman. M. S. and Verma, I. M. Transcriptional activation by BRCA1. Nature expression of BRCA2 protein is also cell cycle regulated, being (Lond.), 382: 678-679, 1996. 11. Sharan. S. K., Morimatsu, M., Albrecht, U.. Lim, D-S., Regel, E.. Dinh, C., Sands. A., induced at late G,/early S phase, before DNA synthesis. Because Eichele. G.. Hasty. P.. and Bradley. A. Embryonic lethality and radiation hypersensitivity BRCA2 has been implicated in DNA repair, its presence during DNA mediated by RadSI in mice lacking Bnu2. Nature (Lond.). 386: 804-810, 1997. synthesis may reflect a role in maintaining genome integrity during 12. Lachmann, P. J., Strangeways. L., Vyakarnam. A., and Evans, G. Raising antibodies by coupling peptides to PPD and immunizing BCG-sensitized animals. 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5488

David Bertwistle, Sally Swift, Nicola J. Marston, et al.

Cancer Res 1997;57:5485-5488.

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