Overexpression of a Protein Fragment of RNA Helicase a Causes Inhibition

Overexpression of a protein fragment of RNA helicase A causes inhibition of endogenous BRCA1 function and defects in ploidy and cytokinesis in mammary epithelial cells

Brian P Schlegel1, Lea M Starita and Jeffrey D. Parvin

Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA

The breast- and ovarian-specific tumor suppressor, Among these proposed activities of BRCA1, it is unclear BRCA1, has been implicated to function in many nuclear which of these are directly dependent upon normal processes, including DNA damage repair, recombination, BRCA1 function, and which are secondary effects. transcription, ubiquitination, cell cycle checkpoint enfor- Mutation of BRCA1 results in a defect in the cement, and centrosome regulation. Utilizing a previously maintenance of genomic stability. HCC1937 cells, tumor described interaction between BRCA1 and RNA helicase cells of breast epithelial origin that have mutated both A (RHA), we have developed a dominant-negative BRCA1 alleles, are sensitive to g-irradiation and approach to block BRCA1 function in human breast defective for transcription-coupled repair (Abbott et al., epithelial cells. Overexpression of a truncated RHA 1999). Studies of BRCA1-deficient mouse embryos and peptide that can bind to the BRCA1 carboxy-terminus derived mouse cell lines reveal hypersensitivity to g- prevents normal BRCA1 function, such as BRCA1 irradiation, numerical and structural chromosomal association with nuclear foci following DNA damage. abnormalities, a defective G2/M checkpoint, aneuploi- Overexpression of this dominant-negative protein induces dy, and defects in the processes of homologous pleomorphic nuclei, aberrant mitoses with extra centro- recombination and transcription-coupled repair (Gowen somes, and tetraploidy. This model system allows us to et al., 1998; Shen et al., 1998; Moynahan et al., 1999; observe changes to mammary epithelial cells that occur Snouwaert et al., 1999; Xu et al., 1999). Also, BRCA1 acutely following loss of BRCA1 function. Furthermore, forms nuclear foci during S phase that disperse and inhibition of BRCA1 via overexpressing the RHA subsequently reform in response to DNA-damaging fragment coincides with a reduction in PARP-1 protein agents (Scully et al., 1997b). The role of BRCA1 in expression, suggesting a possible mechanism for BRCA1 genomic stability appears critical for all cell types, not in the maintenance of genomic integrity. just breast and ovarian epithelial cells, the progenitors of Oncogene (2003) 22, 983–991. doi:10.1038/sj.onc.1206195 BRCA1-dependent tumors. Experiments that alter BRCA1 expression have Keywords: BRCA1; RNA helicase A; aneuploidy; revealed several phenotypes. Overexpression of wild- centrosome; PARP-1 type BRCA1 results in apoptosis following induction of GADD45 and activation of the JNK pathway (Harkin et al., 1999). Other experiments in which the wild-type BRCA1 gene is overexpressed have resulted in slowed Introduction progression through the cell cycle, by induction of p21 or the GADD45 and GADD153 genes (Somasundaram The function of the breast- and ovarian-specific tumor et al., 1997; MacLachlan et al., 2000). It is unclear if suppressor, BRCA1, has been analysed in several these phenotypic changes are a direct result of altered systems. The various functions ascribed to BRCA1 BRCA1 function. Acute inhibition of BRCA1 may include transcription (Monteiro et al., 1996; Scully et al., permit the distinction between primary and secondary 1997a), chromatin remodeling (Hu et al., 1999; effects. Bochar et al., 2000), cell cycle checkpoint enforcement Inhibition of BRCA1 function in human breast (Larson et al., 1997; Somasundaram et al., 1997; epithelial cells would provide a model system to MacLachlan et al., 2000), various pathways of DNA investigate the molecular basis for BRCA1-dependent repair (Gowen et al.,1998;Abbottet al., 1999; tumorigenesis. Towards this end, we have employed a Moynahan et al., 1999; Snouwaert et al.,1999),and new method to block normal BRCA1 function by centrosome replication (Xu et al., 1999; Hsu et al.,2001). overexpressing a protein that binds to the carboxy terminus of BRCA1. Of the cancer-predisposing muta- *Correspondence: JD Parvin; E-mail: [email protected] tions in BRCA1, 90% disrupt the carboxy-terminal 1Current address: Department of Pathology, University of Illinois at domain (Szabo and King, 1995), highlighting the Chicago, Chicago, IL 60607, USA Received 18 June 2002; revised 22 October 2002; accepted 30 importance of this domain in the tumor-suppressor October 2002 function of BRCA1. BRCA1 fused to the GAL4 Inhibition of BRCA1 by a dominant-negative RHA BP Schlegel et al 984 DNA-binding domain activates transcription in vitro on dependent upon this domain. MCF10A cells were promoters containing GAL4 sites (Haile and Parvin, infected with virus expressing LacZ or DN-RHA at a 1999; Schlegel et al., 2000). Overexpression of an multiplicity of infection (MOI) of 350 plaque-forming RHA(1–344) peptide containing the BRCA1-binding units (PFU)/cell, and harvested 27 h postinfection (hpi). domain inhibits GAL4-BRCA1-dependent transcrip- At 27 hpi, morphological changes in MCF10A cells are tional activation from a GAL4 site-dependent reporter apparent (see below). At 48 hpi, for both MCF10A and in transiently transfected HeLa cells, indicating that U2OS cells, there was complete cell death only in those RHA can regulate BRCA1 function in human cells cultures that express DN-RHA. The cause of DN-RHA- (Anderson et al., 1998). induced cell death at 48 hpi remains to be determined. In this study, we inhibit the function of BRCA1 in Immunoprecipitation of DN-RHA from 27 hpi extracts normal mammary epithelial cells and identify the of MCF10A cells using an antibody directed against its consequences of BRCA1-inhibition. We overexpress a FLAG epitope also purified BRCA1, demonstrating peptide containing the BRCA1-binding domain of that DN-RHA interacts with endogenous BRCA1 RHA, which lacks other functional RHA domains, in (Figure 1c). The immunoprecipitation was specific since order to inhibit the normal function of endogenous incubation of the cell lysate with a competing FLAG BRCA1. MCF10A cells are particularly well suited for peptide prevented immunoprecipitation of both DN- this study, since they are a near-diploid nontumorigenic RHA and BRCA1. human breast epithelial cell line. Although we find that this strategy does indeed inhibit BRCA1 function in both MCF10A cells and an osteosarcoma cell line, we find that only in MCF10A cells do we observe changes Inhibition of BRCA1 nuclear distribution by DN-RHA consistent with early lesions in breast cancer. Expression of DN-RHA altered the normal nuclear distribution of BRCA1 in response to DNA damage. Treatment of MCF10A cells or U2OS cells with Results bleomycin caused dispersal of BRCA1 nuclear foci within 1 h, with reappearance of foci containing BRCA1 Strategy for inhibition of BRCA1 after 6 h (Figure 2). Viral titers used to infect MCF10A (350 PFU/cell) and U2OS (100 PFU/cell) cells were Inhibition of endogenous BRCA1 function was achieved normalized according to expression of the DN-RHA by overexpression of a fragment of the RHA protein polypeptide. Infection of MCF10A or U2OS cells with that binds to the carboxy-terminus of BRCA1. Im- control virus does not significantly alter BRCA1- munoprecipitation of endogenous RHA using cell lysate containing focus formation before or after DNA from U2OS cells co-precipitates endogenous BRCA1 damage. By contrast, prior infection of MCF10A and (Figure 1a), demonstrating that RHA and BRCA1 U2OS cells with the DN-RHA virus markedly reduces associate in vivo. We hypothesize that overexpression of reformation of BRCA1-containing nuclear foci 6 h after a fragment of the RHA protein, which binds only to DNA damage in both cell lines (Figure 2). Since DN- BRCA1 yet lacks other key domains of the RHA RHA binds endogenous BRCA1 and changes the polypeptide, will function as a dominant-negative response of BRCA1 to DNA damage, expression of inhibitor of BRCA1 function (Figure 1b). The region DN-RHA thus alters normal BRCA1 function and can of RHA-containing residues 89–344 includes sequences be used to identify acute phenotypic and molecular that were parts of domains shown to bind to CBP changes associated with the loss of BRCA1 function. (residues 1–250) and RNA polymerase II (residues 230– 650)(Nakajima et al., 1997). We have found that RHA residues 1–88 are sufficient to bind to CBP (unpublished observations), but the RHA(89–344) protein fused to Overexpression of DN-RHA causes changes in nuclear glutathione S-transferase bound to CBP and RNA morphology polymerase II at low levels (data not shown). Thus, while this strategy is targeted to affect endogenous Common features of BRCA1-associated tumors are BRCA1 function, it may also affect CBP and polymer- pleomorphic nuclei and aneuploidy. MCF10A cells ase activities. Arguing against a broad effect of this infected with the DN-RHA virus formed distinctive treatment on CBP and RNA polymerase II function, bilobed nuclei (Figure 3). Mock-infected or control cells overexpressing the dominant-negative RHA(89–344) virus-infected MCF10A cells exhibited no change in peptide exhibited few changes in gene expression by nuclear morphology. In contrast, although U2OS cells microarray analysis (data not shown). exhibited irregular nuclear morphology in response to Human breast epithelial MCF10A cells, or U2OS infection with either the control or DN-RHA virus, few osteosarcoma cells, were infected with a recombinant cells formed the distinctive bilobed nuclei seen in the adenovirus that acutely expresses the dominant-negative mammary cell line expressing DN-RHA (Figure 3). A RHA(89–344) peptide (DN-RHA) or a control virus that nuclear membrane could be detected by phase micro- expresses LacZ. Overexpressed DN-RHA will compete scopy separating these nuclei within single MCF-10A with endogenous proteins binding to the BRCA1 cells (data not shown), and such cells have been termed carboxy-terminus and thus block BRCA1 functions binuclear.

Oncogene Inhibition of BRCA1 by a dominant-negative RHA BP Schlegel et al 985

Figure 1 Approach to inhibition of BRCA1 function. (a) Coimmunoprecipitation of endogenous RHA and BRCA1. RHA was precipitated from U2OS cell lysate using preimmune serum (lane 3) or a serum from a rabbit immunized against amino acids 1–250 of RHA (lane 4). Lanes 1 and 2 represent 2% of input. (b) Design of dominant-negative inhibitor. Top: RHA contains a distinct BRCA1- binding domain, independent of helicase functions (Nakajima et al., 1997; Anderson et al., 1998). Middle: Map of the dominant- negative RHA peptide (DN-RHA). DN-RHA consists of amino acid residues 89–344 of the RHA primary sequence, which includes the BRCA1-binding domain, fused to a nuclear localization sequence (NLS) and a FLAG epitope. Bottom: Schematic representation of the interaction between DN-RHA and BRCA1. BRCA1 contains two structural motifs, an N-terminal RING ﬁnger domain, which is implicated in ubiquitin ligation, and a carboxy-terminal tandem BRCT repeat, implicated in transcriptional activation and protein– protein interactions. DN-RHA binds to the carboxy-terminal domain of BRCA1 and inhibits the transcriptional activation function. (c) Coimmunoprecipitation of DN-RHA and endogenous BRCA1. MCF10A cells were infected with the DN-RHA virus or control virus, and cells were lysed 27 hpi. DN-RHA was precipitated using an antibody directed against the FLAG epitope, in the absence or presence of a competing FLAG peptide (0.2 mg/ml). Lanes 1–3 represent 2% of input

Overexpression of DN-RHA results in tetraploidy in 27 hpi cells were fixed and stained with propidium iodide MCF-10A cells (Figure 4a (left column), and b). For comparison, U2OS cells were infected such that DN-RHA expression was Since binuclear cells may reflect a failure to complete the same as in MCF10A cells, at an MOI of 100 PFU/ mitosis, we measured the DNA content of MCF10A cell (Figure 4a (middle column), and c), or with the same and U2OS cells infected with DN-RHA and control virus load as the MCF10A cells (Figure 4a, right virus by flow cytometric analysis. MCF10A cells were column). In no case was a significant change in the infected with virus expressing LacZ or DN-RHA, and at G1,S,orG2 population observed for U2OS cells. By

Oncogene Inhibition of BRCA1 by a dominant-negative RHA BP Schlegel et al 986 a

MCF10A

U2OS

mock control DN-RHA

b 80 70 MCF10A 60 mock 50 GFP 40 DN-RHA

%cells 30 20 10 0 0 hour 1 hour 6 hours c 80 70 U2OS 60 50 mock 40 GFP

%cells 30 DN-RHA 20 10 0 0 hour 1 hour 6 hours

Figure 2 Expression of DN-RHA alters reformation of nuclear foci containing BRCA1 in response to DNA damage. MCF10A or U2OS cells were infected with adenovirus expressing GFP or DN-RHA. Cells, 24 h postinfection, were treated for 30 min with 100 mg/ ml bleomycin and 6 h later the cells were ﬁxed, permeabilized, and stained using an antibody directed against BRCA1. Cells were stained with DAPI to visualize DNA (blue). BRCA1 staining is shown in red. (a) Fluorescence micrographs of MCF10A (top) and U2OS (bottom) cells, uninfected (left), infected with the control virus (middle), or the DN-RHA virus (right). (b) Bar graph indicating the percentage of MCF10A cells exhibiting nuclear foci containing BRCA1 after infection with the DN-RHA virus or control virus and bleomycin treatment. (c) Same as (b) except using U2OS cells

contrast, MCF10A cells exhibited a tetraploid popula- inhibition of BRCA1 function by the expression of the tion only in response to DN-RHA expression (Figure DN-RHA was associated with centrosome amplifica- 4a, b). U2OS cells did not exhibit tetraploidy, even when tion. Uninfected MCF10A cells, stained with DAPI to a large excess of virus was used during the infection visualize DNA, showed normal interphase nuclei, and (Figure 4 and data not shown). staining for g-tubulin revealed normal centrosome Aneuploidy is often accompanied by abnormal number (Figure 5a, Table 1). All mitotic cells contained centrosome amplification. In addition, centrosome two separated centrosomes and condensed DNA amplification is seen in early lesions in breast cancer (Figure 5b). Cells infected with a control virus were (Lingle et al., 2002). We thus tested whether the similar (Table 1). By contrast, MCF10A cells infected

Oncogene Inhibition of BRCA1 by a dominant-negative RHA BP Schlegel et al 987 a b c

MCF10A

d e f

U2OS

mock LacZ DN-RHA

Figure 3 Dominant-negative inhibition of BRCA1 produces binuclear MCF10A cells. MCF10A (a–c) or U2OS cells (d–f) were infected with the DN-RHA virus or control virus, ﬁxed 27 hpi, and stained with DAPI to visualize DNA. Cells were uninfected (a, d), infected with the LacZ-expressing adenovirus (b, e), or with the DN-RHA-expressing adenovirus (c, f)

with the DN-RHA virus were binuclear, as shown the DN-RHA expression inhibits BRCA1 function. earlier and had extra centrosomes in interphase cells HCC1937 cells contain enlarged nuclei, indicative of (Figure 5c). Although the total number of mitotic cells increased DNA content, numerous centrosomes, and was unaffected, half of the mitotic cells contained four are binuclear. The karyotype of HCC1937 cells indicates centrosomes and exhibited unequal chromosome segre- that the cells are approximately tetraploid (Tomlinson gation (Figure 5d, e; Table 1). Four centrosomes could et al., 1998). The exaggerated morphological features of result from a second cell cycle after a failed cell division, HCC1937 cells (Table 2) were strikingly similar to the but we suggest that this possibility was unlikely since the phenotype of MCF10A cells with BRCA1 inhibited by timing was too rapid. In other experimental systems, the DN-RHA expression. Since these features were appar- accumulation of extra centrosomes after a failed cell ent within 27 hpi in MCF10A cells, binuclear morphol- division required 40 h (Meraldi et al., 1999), but we ogy, aberrant centrosome amplification, and tetraploidy observed the extra centrosomes in a high fraction of the represented the acute changes that occur upon loss of mitotic cells after 27 h, approximately the length of time BRCA1 function. of a single cell cycle for MCF10A cells. U2OS cells did not exhibit either feature of extra centrosomes or of PARP-1 expression is decreased in DN-RHA expressing abnormal mitoses. Quantitation of the morphological mammary cells changes in MCF10A and U2OS cells in response to inhibition of BRCA1 is summarized in Table 1. Inter- The phenotype that results from inhibition of BRCA1 estingly, the percentage of MCF10A cells exhibiting function in MCF10A cells was reminiscent of cells that binuclear morphology (22.8%) was roughly consistent lack poly(ADP-ribose) polymerase (PARP-1). PARP-1 with the aneuploid population (16.2%; Figure 4). catalyzes the reversible (ADP-ribosyl)ation of nuclear Examination of BRCA1 mutant HCC1937 cells proteins such as histones, DNA polymerase a and revealed many of the same features as MCF10A cells b, PCNA, p53, and automodification of PARP-1 infected with the DN-RHA virus (Figure 5f–g). (D’Amours et al., 1999). The enzymatic activity is HCC1937 cells are an established cell line with one stimulated upon binding to a single-strand or double- BRCA1 allele containing a carboxy-terminal truncation, strand DNA breaks (Ohgushi et al., 1980; Benjamin and and the other BRCA1 allele is deleted. This cell line is Gill, 1980). PARPÀ/À mouse fibroblasts exhibit abnor- also mutant for p53 and PTEN (Tomlinson et al., 1998). mal nuclear morphology and tetraploidy (Simbulan- Although HCC1937 cells are not directly comparable to Rosenthal et al., 1999, 2000). We examined PARP-1 MCF10A cells acutely overexpressing DN-RHA, a mRNA and protein expression in MCF10A cells comparison of the phenotypes supports the notion that infected with the DN-RHA virus. PARP-1 mRNA

Oncogene Inhibition of BRCA1 by a dominant-negative RHA BP Schlegel et al 988 by DN-RHA expression. The absence of an 89 kDa PARP-1 proteolytic fragment indicated that the drop in PARP-1 protein was not due to caspase cleavage of the full-length 113 kDa protein, one of the initial events of apoptosis (Lazebnik et al., 1994). There was no change in the expression of PARP-1 in U2OS cells, suggesting that BRCA1 regulation of PARP-1 expression may be cell type-speciﬁc.

Discussion

We have exploited the interaction between the BRCA1 carboxy-terminus and RHA to develop a dominant- negative approach, which acutely inhibits BRCA1 function in human breast epithelial cells. This approach permits the evaluation of changes in phenotype that occur soon after loss of BRCA1 function. Overexpres- sion of the DN-RHA peptide elicited a host of phenotypic changes in MCF10A cells consistent with the proposed role of BRCA1 function in the maintenance of genomic stability. These changes include a failure of BRCA1 foci to reform after DNA damage, binuclear morphology, tetraploidy, and abnormal centrosome amplification. We examined a BRCA1-deficient cell line, HCC1937, and discovered similar abnormalities in nuclear morphology and centrosome number, suggesting that loss of BRCA1 function is directly responsible for this phenotype. It was previously unknown that the centrosome number is abnormal in these cells. Prior studies have demonstrated that BRCA1-deficient cells, derived from knockout mouse embryos, develop aneuploidy and extra centrosomes, but this phenotype is evaluated after many days of growth (Shen et al., 1998; Xu et al., 1999). Our ability to induce centrosome expansion within 27 h by acute overexpression of a dominant-negative inhibitor of BRCA1 function suggests that BRCA1 directly regulates the Figure 4 Expression of DN-RHA induces tetraploidy in pathway controlling centrosome replication. Overex- MCF10A cells. Cells were infected with the DN-RHA virus or pression of DN-RHA may relieve this inhibitory control virus and plates were analysed 27 hpi. (a) Histograms of MCF10A and U2OS cells infected with adenovirus expressing function. Perhaps some cell types, such as U2OS cells, LacZ or DN-RHA. MCF10A cells were infected with 350 PFU/cell may have a second pathway regulating centrosome (left column). U2OS cells were infected with 100 PFU/cell (matched replication independent of BRCA1, so that centrosome for protein expression, middle column) or with 350 PFU/cell replication remains normal. BRCA1 has been found to (matched for virus titer, right column). (b) Cell cycle profile of MCF10A cells. DNA content is indicated as 2N (G1 phase), associate with centrosomes in mitotic cells (Hsu and 2oNo4 (S phase), 4N (G2/M phase), or >4N (aneuploid). White, 1998; Hsu et al., 2001). An internal domain of Significant numbers of aneuploid cells were detected only in BRCA1, amino acid residues 504–803, binds directly to MCF10A cells expressing DN-RHA. (c) Cell cycle profile of U2OS g-tubulin, and overexpression of this domain results in cells centrosome expansion in stably transfected COS-7 cells (Hsu et al., 2001). A comparison of our results with other experimental results in which BRCA1 was inhibited is illustrative. In levels dropped by an average of 50% in three experi- experiments in which the isolated carboxy-terminus of ments in response to DN-RHA expression, when BRCA1 was overexpressed, the G2-M cell cycle normalized to GAPDH mRNA (Figure 6a). PARP-1 checkpoint was defective (Larson et al., 1997). In one protein levels decrease much more dramatically set of experiments the BRCA1 concentration was (Figure 6b), indicating that BRCA1 regulates PARP-1 reduced in HBL100 cells using a BRCA1-specific expression at both the transcript and the protein level. ribozyme. Ribozyme-treated cells were sensitive to As an internal control, caspase 3 levels were unaffected DNA-damaging agents and resistant to microtubule-

Oncogene Inhibition of BRCA1 by a dominant-negative RHA BP Schlegel et al 989 interphase mitotic ab MCF10A control

c d e MCF10A DN-RHA

f g h

HCC1937

Figure 5 Loss of BRCA1 function results in abnormal centrosome replication. Cells were stained with DAPI to visualize DNA (blue) and an antibody directed against g-tubulin to visualize centrosomes (red). (a–e) MCF10A cells, (c–e) MCF10A cells infected with the DN-RHA adenovirus, (f–h) BRCA1-mutant HCC1937 cells. Interphase nuclei are in a, c, f, and g. A normal mitosis with condensed chromatin and two centrosomes is in b. Abnormal mitoses with extra centrosomes are in d, e, and h. This phenotype of the BRCA1 mutant cell line is highly similar to the phenotype of the MCF10A cells overexpressing DN-RHA

Table 1 Changes in nuclear morphology upon inhibition of BRCA1 Table 2 Nuclear morphology of HCC1937 cells function Interphase cells Mitotic cells Interphase cells Mitotic cells Simple Enlarged Bi-nuclear Simple nuclei Binuclear Centrosomes nuclei nuclei 24Centrosomes 1 or 2 >2 >2 2 >2

MCF10A 63.2a (153)b 15.5 (40) 18.2 (47) 0.4 (1) 2.7 (7) Mock 94.6a (952)b 3.0 (30) 2.2 (22) 0.2 (2) LacZ 87.5 (851) 9.4 (91) 2.8 (27) 0.3 (3) aPercentage of cells with a given morphology is shown. bActual cell DN-RHA 74.8 (1581) 22.8 (481) 1.2 (26) 1.2 (25) counts in parentheses U2OS Mock 96.7 (963) 2.2 (22) 0.9 (9) 0.1 (1) LacZ 95.6 (932) 1.7 (17) 2.6 (27) 0 mitoses and centrosome accumulation, suggesting that DN-RHA 92.6 (881) 6.6 (63) 2.7 (26) 0 BRCA1 function is important in these processes. The common thread among these four different approaches aPercentage of cells with a given morphology is shown. bActual cell toward inhibition of BRCA1 function lends support to a counts in parentheses role for BRCA1 in the regulation of centrosome replication and mitosis. Our study further adds the interfering agents (Lafarge et al., 2001). When the g- important insight that while BRCA1 nuclear dot tubulin-binding domain of BRCA1 was stably over- redistribution was inhibited by DN-RHA in both the expressed in COS-7 cells, cell death with abnormal U2OS and MCF10A cells, only in the MCF10A cells centrosome ampliﬁcation was observed (Hsu et al., were the mitotic phenotypes apparent. This observation 2001). In our experiments, we also observed abnormal suggests that the effects of BRCA1 may be cell

Oncogene Inhibition of BRCA1 by a dominant-negative RHA BP Schlegel et al 990 a b MCF10A U2OS LacZ DN-RHA mock DN-RHA LacZ mock DN-RHA LacZ PARP 177 114 PARP 81 GAPDH 64 50 12 37 Caspase3 26 15 123 456

Figure 6 Loss of BRCA1 function decreases PARP expression. (a) Northern blot analysis of mRNA from MCF10A cells infected with adenovirus expressing LacZ or DN-RHA. Cells were harvested 27 hpi and mRNA was puriﬁed. mRNA samples from control (lane 1) or DN-RHA (lane 2) virus-infected cells were probed for the PARP-1 gene (top) and for the GAPDH gene (bottom). (b) Immunoblot of MCF10A (lanes 1–3) or U2OS (lanes 4–6) cell lysate from infected cells. Samples were 27 hpi lysates from uninfected (lanes 1, 4), DN-RHA (lanes 2, 5), or LacZ (lanes 3, 6) virus-infected cells

type-specific, and this is consistent with BRCA1 being a Materials and methods breast cancer-specific tumor suppressor. Protein expression and immunoprecipitation A recombinant Inhibition of BRCA1 in MCF10A cells results in a adenovirus expressing a dominant-negative inhibitor of striking decrease in PARP-1 protein expression, BRCA1 function was generated using the Adeno-Quest raising the possibility that many of the observed expression system (Quantum Biotechnologies, Inc.). Virus phenotypic changes may actually be caused by loss of titer was determined by serial dilution using a standard plaque PARP-1. The similarities between BRCA1 and PARP- formation assay. Infected cells were lysed in buffer containing deficient mouse cells are remarkable. Both BRCA1- and 50 mm Tris, pH 7.9, 150 mm NaCl, 1% Nonidet P-40, 5 mm PARP-deficient cells exhibit abnormal nuclear morphol- EDTA, and 0.25% protease inhibitor cocktail (Sigma). ogy, tetraploidy, and hypersensitivity to g-irradiation Supernatant was analysed directly (40 mg) or was incubated 1 (Wang et al., 1995, 1997; de Murcia et al., 1997; overnight at 4 C with M2 agarose (Sigma) or an RHA-directed antibody bound to protein G sepharose (Pharmacia Biotech). Simbulan-Rosenthal et al., 1999, 2000). The observation Samples were washed with lysis buffer (0.1% NP-40) and of chromatid breaks and quadriradial chromosomes resolved by SDS–PAGE, transferred to nitrocellulose, probed (Shen et al., 1998; Xu et al., 1999) has implicated with specific antibody and visualized by chemiluminescence. BRCA1 in sister chromatid exchange (SCE), a form of Fluorescence microscopy Cells were grown on glass cover homologous recombination (Sonoda et al., 1999). slips, fixed and permeabilized as previously described (Scully PARP-deficient mouse fibroblasts exhibit elevated levels et al., 1997b) and washed three times with PBS. Samples were of SCEs (de Murcia et al., 1997; Wang et al., 1997), then incubated for 1 h at 371C with a rabbit polyclonal suggesting that BRCA1 and PARP-1 regulate the antibody directed against BRCA1. Samples were washed three fidelity of SCE. Also, PARP-1 functions as a transcrip- times with PBS, and incubated for 1 h at room temperature tional coactivator (Meisterernst et al., 1997), inhibits with a 1 : 100 dilution of a TRITC-conjugated goat anti-rabbit secondary antibody (Sigma). Samples were washed five times transcription during DNA damage (Taniguchi et al., with PBS, and then mounted on glass slides with a mounting 1982), and enhances transcript elongation (Vispe et al., media containing DAPI (Vectashield). Samples were viewed at 2000), consistent with the observed transcription- Â 1000 magnification using an Axioskop upright microscope coupled repair defect in BRCA1-deficient mouse em- (Carl Zeiss) and images were captured using a model 2.3.1 bryonic stem cells (Abbott et al., 1999; Gowen et al., SPOT digital camera (Diagnostic Instruments, Inc.). For 1998) and with the transcription function of BRCA1 centrosome localization, cells were fixed in methanol followed (Haile and Parvin, 1999; Schlegel et al., 2000). However, by acetone for 10 min at À201C. Staining was performed acute loss of PARP-1 expression is not sufficient to overnight at room temperature using a 1 : 1000 dilution of an account for all roles attributed to BRCA1. Unlike antibody directed against g-tubulin (Sigma). All incubations BRCA1 knockouts, PARP-deficient mice are viable were performed in PBS containing 4% goat serum. Some fluorescent microscopy images were obtained using a Bio-Rad (Wang et al., 1995; de Murcia et al., 1997). Nonetheless, Radiance 2000 confocal and laser scanning microscope identification of PARP-1 as a potential downstream enhanced by a Ti Sapphire Tsunami laser from Spectra- effector of BRCA1 function in the maintenance of Physics. genomic stability represents substantial progress in our Flow cytometry Cells were trypsinized and then washed in understanding of the tumor-suppressor function of PBS containing 1% BSA, fixed overnight in 70% ethanol, BRCA1. incubated with RNAse A (100 mg/ml) and stained for 1 h with

Oncogene Inhibition of BRCA1 by a dominant-negative RHA BP Schlegel et al 991 propidium iodide (50 mg/ml), and then analysed for DNA Acknowledgments content using a Beckman Coulter EPICS XL-MCL flow We thank El Bachir Affar and Stephan Duensing for their cytometer. G2/M cells and tetraploid cells were discriminated helpful advice. This work was supported by Grants RSG-99- from cell doublets using the pulse-width/pulse-area signal 047-04-GMC from the American Cancer Society and NCI ratio. Data were analysed using the Multicycle software Grant CA90281 (JDP), postdoctoral fellowships from the package (Phoenix Flow Systems). Susan G Komen Breast Cancer Foundation, and an NIH Northern analysis mRNA was purified using the FastTrack training grant (BPS) and a predoctoral fellowship from the 2.0 kit. (Invitrogen). Samples were resolved by denaturing Massachusetts Department of Public Health Breast Cancer agarose gel electrophoresis, transferred to a nylon membrane, Research Program (LMS). and hybridized to probe according to standard protocols. Signals were quantified using a PhosphorImager (Molecular Dynamics).

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