We identified IFI16 as a BRCA1-associated protein involved in p53-mediated apoptosis. IFI16 contains the Pyrin/PAAD/DAPIN domain, commonly found in cell death-associated proteins. BRCA1 (aa 502–802) interacted with the IFI16 Pyrin domain (aa 1–130). We found that IFI16 was localized in the nucleoplasm and nucleoli. Clear nucleolar IFI16 localization was not observed in HCC1937 BRCA1 mutant cells, but reintroduction of wild-type BRCA1 restored IFI16 nuclear relocalization following IR (ionizing radiation). Coexpression of IFI16 and BRCA1 enhanced DNA damage-induced apoptosis in mouse embryonic fibroblasts from BRCA1 mutant mice expressing wild-type p53, although mutant IFI16 deficient in binding to BRCA1 did not induce apoptosis. Furthermore, tetracycline-induced IFI16 collaborated in inducing apoptosis when adenovirus p53 was expressed in DNA-damaged p53-deficient EJ cells. These results indicate a BRCA1-IFI16 role in p53-mediated transmission of DNA damage signals and apoptosis.
Germline mutations in the BRCA1 locus predispose women to early-onset, hereditary breast cancer (Miki et al., 1994). Mutations in BRCA1 have been found in approximately 90% of familial breast and ovarian cancers (reviewed by Alberg and Helzlsouer, 1997; Nathanson et al., 2001). BRCA1 encodes a large, multifunctional nuclear phosphoprotein of 1863 amino acids containing the N-terminal RING finger and the C-terminal BRCT domains (Miki et al., 1994; Koonin et al., 1996; Bork et al., 1997).
Recently, mass-spectrometric analysis of BRCA1-interacting proteins revealed the presence of BRCA1-associated genome surveillance complex (BASC) (Wang et al., 2000). This large complex includes ATM/ATR kinases, RAD51, MSH2, MSH6, MLH1, BLM and the RAD50/Mre11/NBS complex, which are all involved in homologous or nonhomologous recombination; colocalization of BRCA1 to these proteins has been demonstrated by immunocytochemical analysis. A possible role of BRCA1 in the DNA repair machinery has also been shown by reintroduction of wild-type or mutant alleles of BRCA1 into the HCC1937 breast cancer cell line, which expresses truncated BRCA1 protein (Scully et al., 1999). Thus, cells into which wild-type BRCA1 has been reintroduced recovered resistance to DNA damage-induced cell death. Significantly, reintroduction of any patient-derived BRCA1 mutants examined has failed to confer resistance to DNA damage on the recipient cells to the same degree as reintroduction of wild-type BRCA1, suggesting that the integrity provided by the complete structure of the BRCA1 protein is necessary for this activity.
A significant body of evidence also indicates that BRCA1 regulates cellular transcription factors such as p53, STAT1 and ZBRK1 (Ouchi et al., 1998; Zhang et al., 1998; Ouchi et al., 2000; Zheng et al., 2000). Functional interaction between BRCA1 and p53 has been shown by murine experiments, where loss of p53 may lead to BRCA1-mutated breast cancer development and regulation of apoptosis (Xu et al., 1999,2001). A role of BRCA1 in gene expression has been further demonstrated by cDNA microarray analysis, in which tetracycline-regulatable BRCA1-induced GADD45 and expression of several cellular genes (Harkin et al., 1999).
IFI16 has been identified as a target of interferon (IFN) α and γ and is a member of the HIN-200 family (reviewed in Johnstone and Trapani, 1999). GAL4DBD-fused full-length IFI16 acts as a potent transcription repressor when positioned in proximity to a promoter containing consensus GAL4DBD binding sequences (Johnstone et al., 1998); however, it is unclear whether IFI16 is involved in chromatin remodeling. Each of the 200 amino acid repeat regions contains such transrepression activity independently, and the N-terminal can bind DNA (Dawson et al., 1995). It has been shown that IFI16 forms stable complexes with cellular transcription factors such as SP1 and p53 (reviewed in Johnstone and Trapani, 1999; Johnstone et al., 2000). More recently, the Pyrin domain, which is commonly found among cell death-associated proteins such as Pyrin, ASC, and zebrafish caspase and is also referred to as the PAAD/DAPIN domain, has been found in N-terminal IFI16, suggesting a role of IFI16 in the apoptotic pathway (reviewed in Aravind et al., 2001; Fairbrother et al., 2001; Martinon et al., 2001; Pawlowski et al., 2001; Staub et al., 2001). Presumably, IFI16 regulates the activity of certain transcription factors in the nucleus that are involved in the commitment to cell death.
In the present study, we further analysed the BRCA1-associated components identified through the mass spectrometric analysis. We identified IFI16 as a previously unknown member of BASC, and found that both IFI16 and BRCA1 colocalize in most of the cells examined by confocal laser microscopic analysis. IFI16 subcellular localization was transiently changed after DNA damage by IR, suggesting that IFI16 is involved in the DNA damage pathway. Interestingly, IFI16 distribution, both in the nucleoplasm and nucleolar, is differentially regulated in normal human epithelial cells after DNA damage, but such regulation was abolished in the BRCA1-mutant breast cancer cell line. Most importantly, nucleolar accumulation of IFI16 was restored by reexpression of wild-type BRCA1 in BRCA1-mutant cells, strongly suggesting that IFI16 is involved in the BRCA1 pathway activated by DNA damage. Finally, studies using mouse embryonic fibroblasts (MEFs) obtained from BRCA1-mutant mice with exon 11 deleted or tetracycline-regulatable IFI16 in EJ cells revealed proapoptosis activity of IFI16 under conditions of DNA damage, supporting a model of tumor suppression in which the BRCA1/IFI16 complex is involved in the p53-mediated cell death pathway.
Identification of specific interaction between BRCA1 and IFI16
Our analysis of proteins coimmunoprecipitating with BRCA1 identified a BRCA1-associated genome surveillance complex (BASC) containing proteins of the DNA damage and DNA repair responses such as MSH2/MSH6, MLH1/PMS2, ATM, BLM, replication factor C (RFC) and the RAD50-MRE11-NBS1 complex as well as p53 and STAT1 transcription factors (Ouchi et al., 1998; Zhang et al., 1998; Ouchi et al., 2000; Wang et al., 2000; Zheng et al., 2000). We identified other proteins with no known function in the DNA damage or DNA repair responses. Among them is the Pyrin domain-containing nuclear protein IFI16 (Trapani et al., 1992; Aravind et al., 2001), which coimmunoprecipitated with BRCA1 when exposed to anti-BRCA1 antibody C20 (Figure 1a).
Interaction of BRCA1 and IFI16 was confirmed by coimmunoprecipitation from normally growing HeLa cell nuclear extracts using a novel mouse monoclonal antibody (MAb21A8, ref. Okada and Ouchi, 2003) against the BRCA1 C-terminal and C20 polyclonal antibody. BRCA1 was immunoprecipitated from 300 μg of nuclear extract and samples immunoblotted with mouse anti-IFI16 monoclonal antibody 1G7. Three major IFI16 products were detected in the HeLa nuclear extract as a result of alternative splicing of the IFI16 mRNA (Johnstone and Trapani, 1999). Both anti-BRCA1 antibodies coimmunoprecipitated endogenous IFI16 (Figure 1b). We tested 12 anti-BRCA1 antibodies, which all resulted in BRCA1 and IFI16 coimmunoprecipitation (data not shown). Reciprocal coimmunoprecipitation confirmed the interaction by immunoprecipitation of IFI16 followed by BRCA1 immunoblot (Figure 1c). Densitometrically, ∼30% of BRCA1 bound to ∼50% of IFI16.
Next, we mapped the BRCA1-IFI16 binding region by means of a GST-pulldown assay using 2 μg purified GST-fusion protein and 500 μg HEK293T total cell lysate. These results showed that BRCA1 amino acids 502–802 bound to the Pyrin domain (aa 1–130) of the IFI16 N-terminal region (Figure 1d,e). Using HEK293T cells, interaction of BRCA1 with the IFI16 N-terminal was confirmed by coimmunoprecipitation with HA-tagged wt but not N-terminal truncated IFI16 (IFI16ΔN) lackingaa 2–169 (Figure 1f). C-terminal-truncated BRCA1 in HCC1937 breast cancer cells bound weakly to IFI16, indicating that the center region interacted with IFI16 (Figure 1g). DNaseI treatment of cell lysates did not affect coimmunoprecipitation, suggesting that the proteins reside in the same complex, rather than interact through their DNA. Thus, BRCA1 physically interacts with IFI16 in normally growing cells.
Localization of IFI16 in mammary epithelial cells
Previous studies demonstrated subcellular BRCA1 localization changes after DNA damage such as that due to UV or IR treatment or hydroxyurea administration (Scully et al., 1997b). We studied whether DNA damage affects IFI16 localization using confocal microscopy. IFI16 and BRCA1 were detected by anti-IFI16 monoclonal antibody 1G7 (green) and anti-BRCA1 polyclonal antibody K-18 (red). Normally, BRCA1 localization in MCF10A cells is dispersed (Figure 2b). BRCA1 signals slightly decreased 30 min after IR (5 Gy) of DNA damage returned to the basal level after 1 h and demonstrated a large dot pattern in 3 h (Figure 2f, j, n). IFI16 was localized in the nucleoplasm and nucleoli (Figure 2c) and IFI16 colocalization with BRCA1 was detected throughout the nuclear within 0.5 h after IR (Figure 2d, h). However, following DNA damage, nucleoplasmic IFI16 decreased within 1 h without significant change in nucleolar distribution (Figure 2k), leading to reduced nucleoplasm colocalization with BRCA1 shown by yellow signals in merged images (Figure 2l). Nucleoplasmic IFI16 reappeared after 3 h and colocalization with BRCA1 was detected as yellow signals where BRCA1 forms a large dot pattern (Figure 2n–p). As shown in Figure 2q, nucleolar localization of IFI16 (green) was confirmed by staining untreated MCF10A cells with antinucleolin (red). IFI16 Western blotting and coimmunoprecipitation indicated transient decrease in BRCA1 expression after IR, with return to original levels after 1 h (Figure 2r, s). Thus, DNA damage determines IFI16 localization in nuclei.
Wild-type BRCA1 is required for relocalization of IFI16 after IR
To further study IFI16's role in BRCA1-associated breast cancer, we explored its localization in the BRCA1 mutant HCC1937 human breast cancer cell line and wt-BRCA1-re-expressing HCC1937 cells (Tomlinson et al., 1998; Scully et al., 1999). IFI16 nucleolar localization was not obvious in parental HCC1937 cells, but a clear nucleolar pattern was detected in wt-BRCA1 reexpressing HCC1937 cells (Figure 3b, B). IFI16 and mutated BRCA1 redistribution in parental HCC1937 cells was not observed 3 h after IR (Figure 3f–o). Significantly, in wt-BRCA1-reexpressing HCC1937 cells, IFI16 nucleolar localization increased 1 h after DNA damage with nucleoplasm redistribution within 3 h (Figure 3J, N), a pattern like that in IR-treated MCF10A cells (Figure 2k, o). Reintroduced BRCA1 showed the large dot pattern 3 h after treatment (Figure 3o). As shown in MCF10A cells, colocalization of both proteins was detected as yellow signals where BRCA1 forms a large dot pattern (Figure 3p). These results strongly imply IFI16's involvement in the BRCA1 DNA damage pathway and the requirement of wt-BRCA1 protein for its redistribution.
IFI16 induces apoptosis in BRCA1- and p53-positive cells
The IFI16 effect in genotoxic stress-mediated apoptosis was examined using BRCA1(−) MEFs according to a published protocol (Cortez et al., 1999; Xu et al., 2002). BRCA1(−)/p53(−) MEFs (Xu et al., 2001) were stably infected with wt-p53-expressing retrovirus or vector alone, yielding BRCA1(−)/p53(−) and BRCA1(−)/p53(+) MEFs (Figure 4a, upper panel). These MEFs were infected with adenovirus for 24 h and treated with IR (5 Gy). IFI16 and BRCA1 were similarly expressed in coinfected MEFs, resulting in expression levels of these proteins similar to those of MCF10A normal epithelial cells (Figure 4a, upper and lower panels). Cell viability was determined by trypan blue exclusion. Neither significant apoptosis nor inhibition of cell growth was detected in BRCA1(−)/p53(−) MEFs and IR-untreated BRCA1(−)/p53(+) MEFs after expressing the indicated proteins (Figure 4b–d). In contrast, BRCA1(−)/p53(+) MEFs varied phenotypes after IR treatment according to the proteins transduced (Figure 4e): Expression of LacZ-, IFI16- or IFI16ΔN deficient in binding to BRCA1 slightly retarded cell growth, although no obvious apoptosis was detected. Furthermore, expression of BRCA1 alone or BRCA1 plus IFI16ΔN resulted in resistance to growth retardation induced by IR treatment. Importantly, coexpression of BRCA1 and IFI16 in BRCA1(−)/p53(+) MEFs led to severe retardation of cell growth and loss of cell viability 36–48 h after IR treatment. These results suggest that IFI16 plays roles in negative regulation of cell growth under conditions of DNA damage when BRCA1 and p53 are coexpressed.
IFI16 collaborates with p53 to induce apoptosis
We further explored the mechanism of apoptosis using tetracycline (tet)-regulated IFI16 in p53-negative human bladder EJ cells (EJ-IFI16), in which IFI16 was induced by removing tet from cell culture (Figure 5a). When tet was removed, cells were infected with the adenovirus indicated in Figure 5b for 24 h. Cells were then treated with actinomycin D (ActD) for 12 h before FACS analysis. Without IFI16 induction (tet(+)), infection with GFP or p53 did not induce apoptosis, which was measured on the basis of the subG1 fraction. When IFI16 was induced (tet(−)), p53 infection strongly induced apoptosis after ActD treatment although p53 alone did not lead to an increased subG1 fraction. These results indicate that IFI16 collaborates with p53 activated by a genotoxic reagent to induce apoptosis.
Mass spectrometric analysis is a well-established technique for identifying specific protein–protein interaction. Previously, we identified several DNA damage repair proteins in BASC – MSH2, MSH6, MLH1, ATM, BLM and the RAD50-Mre11-NBS1 complex (Wang et al., 2000). However, BRCA1 protein has been found to reside in a large protein complex with >2 MDa strongly suggesting that BASC contains many other components. We added IFI16 as a new member to the set of BRCA1-interacting proteins; the specific interaction between BRCA1 and IFI16 was revealed by coimmunoprecipitation experiments. The present data showing the redistribution of IFI16 after DNA damage and the induction of apoptosis in cells with DNA damage suggest that IFI16 is a functional component of BASC. Furthermore, we discovered proapoptosis activity of IFI16 in cells expressing both wild-type p53 and BRCA1.
Recent studies applying a profile-to-profile alignment algorithm to a library of apoptosis-associated proteins identified a novel motif called the Pyrin/PAAD/DAPIN domain (reviewed in Aravind et al., 2001; Fairbrother et al., 2001; Martinon et al., 2001; Pawlowski et al., 2001; Staub et al., 2001). This new class of death domain is found in Pyrin, zebrafish caspase, ASC (apoptosis spec protein), IFI16 and AIM2, suggesting that the proteins in question are involved in cell death pathway. Our studies provided evidence that the Pyrin domain of IFI16 is a protein–protein interaction module that is important for binding to BRCA1.
Confocal laser microscopic images revealed that IFI16 coexists with BRCA1 predominantly in the nuclei in normal mammary epithelial cells. Similar IFI16 localization was observed in human normal diploid fibroblasts (data not shown). Interestingly, only nucleoplasmic IFI16 dramatically decreased 1 h after damage in MCF10A cells whereas the concentration of nucleolar IFI16 was unaffected. Since FACS analysis revealed that the cell cycle profile showed neither cell cycle arrest nor apoptosis within 1 h after IR (data not shown), it is unlikely that relocalization of IFI16 results from cell cycle arrest or apoptosis. Although the mechanism by which nuclear IFI16 localization is regulated remains to be clarified, it is conceivable that DNA damage-activated kinases such as ATM, ATR, Chk2 and DNA-PK play a role in determining IFI16 distribution. IFI16 contains at least four potential phosphorylation consensus motifs (S/TQ) for ATM kinase, namely, at positions 153, 239, 241 and 628.
HCC1937 breast cancer cells demonstrated a different pattern of IFI16 relocalization after DNA damage. Thus, although IFI16 was nuclear in HCC1937 cells, no nucleolar accumulation was observed. The requirement of BRCA1 for IFI16 relocalization after DNA damage was demonstrated in wild-type BRCA1-reintroduced HCC1937 cells. Considering that mutant BRCA1 in HCC1937 cells is differently phosphorylated under conditions of DNA damage than the wild-type protein (Okada and Ouchi, 2003), the BRCA1 phosphorylation status may play a role in determining the localization of the complex containing BRCA1-associated proteins.
It has been shown that BRCA1 increases p53 stability in a p19ARF-dependent manner (Ouchi, T, unpublished observation; ref. Somasundaram et al., 1999). In our investigation of whether IFI16 also affects the p53 protein level, no significant increase was observed in immunoblot analysis (data not shown). The mechanism of apoptosis induced by IFI16 is currently under investigation. In this connection, it is striking that AIM2, one of the proteins containing the Pyrin/PAAD/DAPIN domain (Aravind et al., 2001), is a tumor suppressor protein of human melanoma (Absent In Melanoma, DeYoung et al., 1997). Among proteins containing this domain, loss of apoptosis regulation by the Pyrin domain is likely to be closely associated with tumor malignancy.
Materials and methods
Cell culture and preparation of total cell and nuclear extracts
MCF10A and HCC1937 cells were obtained from ATCC. BRCA1-re-expressed HCC1937 cells were described previously (Scully et al., 1999). Tetracycline-regulatable IFI16 in EJ human bladder carcinoma cells were established following published protocols (Lee et al., 2000). Cells were cultured in DMEM-10% fetal bovine serum (FBS), or RPMI1640-10% FBS (HCC1937 cells). EJ-tet cells were maintained in the presence of tetracycline (1 μg/ml). Cell extracts were prepared in EBC buffer (50 mM Tris, pH 8, 120 mM NaCl, 0.5% Nonidet P-40 [NP-40]), with the addition of 50 mM NaF, 1 mM sodium orthovanadate, 100 μg/ml polymethylsulfonyl fluoride (PMSF), 20 μg/ml aprotinin and 10 μg/ml leupeptin. Nuclear extracts of growing HeLa cells were prepared following a published protocol (Gu et al., 1999). Ionizing radiation was administrated by means of MARK2 IRRADIATOR (JL Stephen & Associate), and actinomycin D (ActD) was purchased from Sigma.
Construction of BRCA1 and IFI16 expression vectors
GST fusion proteins expressing six segments covering full-length BRCA1 have been described (Scully et al., 1997a). Full-length IFI16 cDNA was PCR amplified using following primers: (#1) 5′-IndexTermIndexTermAAAGGATCCATGTCTGTAAAGATGGGAAAAAAATAC-3′ and (#6) 5′-IndexTermIndexTermAGCGGCCGCTTAGAAGAAAAAGTCTGGTGAAGTTTC-3′ from a HeLa cells cDNA library (a gift from Dr H Nojima, Osaka University, Japan). Three segments of IFI 16 cDNAs were PCR amplified by primer (#1) and primer (#2) 5′-IndexTermIndexTermCTGAGCTCCAGGAGTTGCCTCTGCTCC-3′, primer (#3) 5′-IndexTermIndexTermAAAGGATCCAGAAATGTTCTCAAAAAGCCCAGTGATA-3′ and primer (#4) 5′-IndexTermIndexTermAGCGGCCGCTGACATCTGGTTCTTTTTTCTAAGTCG-3′, primer (#5) 5′-IndexTermIndexTermAAAGGATCCAATGACCCCAAGAGCATGAAGCTACCCCAG-3′ and primer (#6). BamHI–SacI or BamHI–NotI fragments amplified by these primers were subcloned into pCRScript (Stratagene), and the DNA sequence was confirmed. Amplified fragments were transferred to pGEX4T vector (Pharmacia). Synthesis of GST fusion proteins and their partial purification on glutathione-sepharose beads were performed as described (Kaelin et al., 1991).
Generation of adenovirus
In this protocol (Ouchi et al., 2000), briefly, cDNAs of GFP, IFI16 and p53 was subcloned into pShuttle-CMV, and recombination with adenovirus genomic DNA was carried out in BJ5180 bacterial cells. Isolated recombinant viruses were amplified in HEK293 cells and used for infection of cells at multiplicity of infection (m.o.i.) of 5 or 50, respectively. Adenovirus BRCA1 was described previously (Ouchi et al., 2000).
Western blot analysis, immunoprecipitation and GST-pulldown assay
Whole cell extract (20 μg) was loaded per lane by 6% SDS–polyacrylamide gel electrophoresis (SDS–PAGE). Transfer onto nitrocellulose was performed using a semidry transfer method (TRANS-BLOT, BIO-RAD), in 50 mM Tris base, 40 mM glycine, 0.37 g/l SDS, 20% methanol (for 3 h at 15 V). After blocking with 1% nonfat dried milk in TBS-T (20 mM Tris, pH 8, 0.9% NaCl, 0.05% Tween-20), the primary antibody, Ab-1 for BRCA1 (Oncogene Science) or 1G7 for IFI16 (Santa Cruz), was used at 2 μg/ml in PBS/1% nonfat dried milk, for 1 h at room temperature. The secondary antibody was peroxidase-conjugated goat anti-mouse IgG (H+L, Jackson Immunoresearch), at 1 : 10 000 in 1% nonfat milk/TBS-T. Signals were developed by ECL (Amersham). IP of BRCA1 was performed as described (Ouchi et al., 2000). Monoclonal antibody 21A8 was generated by immunizing mice with GST-BRCA1 (aa l314–1863) as described (Okada and Ouchi, 2003). The GST-pulldown assay was performed using total cell lysates of HEK293 cells as described (Ouchi et al., 1998,2000).
Cells were fixed for 1 h in phosphate-buffered saline (PBS)-3% paraformaldehyde/2% sucrose solution, followed by 5 min permeabilization at room temperature in Triton buffer (0.5% Triton X-100, 20 mM HEPES, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose). For blocking 5% normal horse serum/5% normal goat serum was used. BRCA1, IFI16 and nucleolin were visualized using rabbit polyclonal antibody K-18, monoclonal 1G7 antibody and rabbit polyclonal antibodoy C23 (all from Santa Cruz), respectively. All secondary antibodies used were species-specific fluorochrome-conjugated antibodies from Jackson Immunoresearch (Texas Red-X for mouse IgG and fluorescein isothiocyanate [FITC] for rabbit IgG), used at 1 : 100 throughout. Nuclei were stained with DAPI. All images were collected by the Leica TCS-SP Confocal Laser Scanning Microscope and processed using Adobe Photoshop software.
Flow cytometry and cell survival assay
For cell cycle analysis of EJ-tetIFI16 cells, tet-removal cells were infected with adenovirus for 24 h and treated with ActD for 12 h. Cells undergoing apoptosis were analysed using FACSCalibur (Beckton and Dickinson). MEFs were described elsewhere (Xu et al., 2001). Stable lines expressing human p53 were generated by infection of MEFs (passage number 1) with retrovirus produced by pBabepuro carrying p53 cDNA and BOSC23 packaging cells (Pear et al., 1993) after puromycin selection (7 days, 1.5 μg/ml). These MEFs (2 × 105) were further infected with the indicated adenoviruses, with or without IR (5 Gy). Cell survival assay using BRCA1-mutant MEFs were studied by trypan blue staining using a published protocol (Cortez et al., 1999; Xu et al., 2002).
BRCA1-associated genome surveillance complex
mouse embryonic fibroblast
human embryonic kidney 293
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We are grateful to Drs David M Livingston and George R Stark for helpful discussion, Ralph Scully for HCC1937-BRCA1 cells, Mutsuko Ouchi for generating and characterizing anti-BRCA1 monoclonal antibodies, Dr Scott Henderson, Jane Sue and Bryan Kloos for confocal laser microscope analysis, and the Mount Sinai School of Medicine Core Facilities for the monoclonal antibody, DNA sequencing and microscopic analysis. This work was supported by an NIH Predoctoral Training Award (JAA), the New York City Council Speaker's Fund (TO), an EMPIRE Grant of State of New York (TO) and National Cancer Institute Awards CA80058, CA78356 (SWL), CA84199 (JQ) and CA79892 and CA90631 (TO).
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