BRCA1 Shifts P53-Mediated Cellular Outcomes Towards Irreversible Growth Arrest

BRCA1 shifts p53-mediated cellular outcomes towards irreversible growth arrest

Pat P Ongusaha1,5, Toru Ouchi2,5, Kyung-tae Kim1, Emily Nytko1, Jennifer C Kwak1, Rosemary B Duda3, Chu-Xia Deng4 and Sam W Lee*,1

1Cancer Biology Program, Hematology/Oncology Division, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02115, USA; 2Derald H Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY 10029, USA; 3Department of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02115, USA; 4Genetics of Development and Disease Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA

The tumor suppressor protein BRCA1 has been shown to or senescence. BRCA1 is also a tumor suppressor gene enhance p53 transcription, whereas activated p53 re- and a number of observations have implicated it in the presses BRCA1 transcription. To further understand the cellular response to DNA damage (Scully et al., 1997; functional interaction of these proteins, we investigated Cortez et al., 1999; Lee et al., 2000; Li et al., 2000; Scully the role of BRCA1 in p53-induced phenotypes. We found and Livingston, 2000; Tibbetts et al., 2000; Wang et al., that BRCA1 when subjected to forced expression acts 2000). Following DNA damage, BRCA1 becomes synergistically with wild-type p53, resulting in irreversible hyperphosphorylated by ATM (Cortez et al., 1999) growth arrest, as shown byVhD mouse fibroblast cells and hCds1/chk2 (Lee et al., 2000) and relocalizes to expressing a temperature-sensitive mutant of p53. complexes containing PCNA (Scully et al., 1997). In Furthermore, reintroduction of both BRCA1 and p53 into addition, BRCA1 plays an important role in transcrip- BRCA1(À/À)/p53(À/À) mouse embryonic fibroblasts tion-coupled repair and in control of cell cycle arrest markedlyincreased the senescence phenotypecompared following DNA damage (Abbott et al., 1999; Scully and to that induced byp53 alone. In particular, we found that Livingston, 2000; Wang et al., 2000). Like p53, BRCA1 BRCA1 expression attenuated p53-mediated cell death in also has an important role in maintaining genomic response to c-irradiation. Moreover, microarrayscreening integrity (Scully and Livingston, 2000). This is strongly of 11 000 murine genes demonstrated that a set of genes supported by data showing that murine embryos upregulated byp53 is enhanced bycoexpression of carrying a BRCA1 null mutation exhibit hypersensitiv- BRCA1 and p53, suggesting that BRCA1 and p53 exert ity to DNA damage and chromosomal abnormalities, a promoter selectivityleading to a specific phenotype. probably because of defective G2/M checkpoint Taken together, our results provide evidence that BRCA1 control and improper centrosome duplication (Scully is involved in p53-mediated growth suppression rather et al., 1999; Somasundaram et al., 1999; MacLachlan than apoptosis. et al., 2000a, b). BRCA1 nullizygous mice show Oncogene (2003) 22, 3749–3758. doi:10.1038/sj.onc.1206439 embryonic lethality in early stage of development associated with a proliferation deficit (Gowen et al., Keywords: BRCA1; P53; Senescence; Microarray; 1996: Hakem et al., 1996; Liu et al., 1996; Xu et al., DNA damage 1999, 2001). BRCA1 nullizygotes also show sponta- neous abnormality of chromosome structure with an increased level of p21/WAF1 in the absence of extrinsic DNA damage. Introduction A BRCA1 exon11 knockout (Brca1D11D11)embryodied late in gestation as a result of widespread apoptosis, but The p53 tumor suppressor gene, which plays a crucial elimination of one p53 allele rescued this embryonic role in cancer progression, is inactivated in most human lethality (Xu et al., 1999). Although the mice were viable, tumors (Levine, 1997; Vogelstein et al., 2000). Depend- most female Brca1D11D11p53+/À mice developed mam- ing on cell context, wild-type (wt) p53 limits cellular mary tumors with loss of the remaining p53 allele within proliferation in response to DNA damage and other 6–12 months (Xu et al., 1999). These studies also clearly cellular stresses by inducing cell cycle arrest, apoptosis, showed that the p53 protein regulates BRCA1 transcription both in vitro and in vivo, and that BRCA1 *Correspondence: SW Lee, Harvard Institutes of Medicine, Room participates in p53 accumulation after DNA damage 921, 4 Blackfan Circle, Boston, MA 02115, USA; through regulation of its phosphorylation and mdm2 E-mail: [email protected] expression (Somasundaram et al., 1997; Ouchi et al., 1998; 5Contributed equally to the work Received 5 November 2002; revised 27 January 2003; accepted 30 Zhang et al., 1998; MacLachlan et al., 2000a, b). Thus, January 2003 several new lines of evidence indicate that loss of BRCA1 BRCA1 effects on p53 function PP Ongusaha et al 3750 is not sufficient for malignant transformation but rather Results triggers multiple genetic alterations, including inactivation of p53 and activation of a number of oncogenes that Collaborative effects between BRCA1 and p53 on ultimately result in mammary tumorigenesis (Brodie et al., expression of p53-target genes and growth arrest 2001; Xu et al., 2001; Venkitaraman, 2002). A significant body of evidence implicates BRCA1 in To explore the biological significance of BRCA1 the regulation of transcription (Chapman and Verma, repression by p53, we first generated stable BRCA1 1996; Monteiro et al., 1996; Somasundaram et al., 1997; transfectants in VhD cells, which contain p53 in a val135) (Wu et al., 1993) Ouchi et al., 1998). BRCA1 has an N-terminal ring temperature-sensitive form (p53 and in p53-null fibroblasts of the 10.1 line, and then finger domain and C-terminal transcription activation determined proliferation at nonpermissive (381C) and domain that activates transcription when fused to a permissive (321C) temperatures for p53 activity. VhD DNA-binding domain (Chapman and Verma, 1996; cells were transfected with a BRCA1 expression plasmid Monteiro et al., 1996). Breast cancer-associated muta- (pcDNA3-BRCA1) or vector alone (pcDNA3) and tions in the BRCT domain abolish this activity, selected with G418 at 381C to avoid p53 activation. supporting the hypothesis that transcriptional regula- Consistent with previous results, a temperature shift to tion by BRCA1 is crucial for tumor suppression. 321C in control cells (VhD-pcDNA3) resulted in a Notably, the BRCT domain binds to several transcription activators or repressors including CBP/p300, CtIP/ significant reduction of endogenous BRCA1 mRNA expression while known p53-target genes were induced CtBP, and histone deacetylases, HDAC (Deng and following p53 activation (Figure 1a). However, VhD Brodie, 2000). Previous studies have shown that BRCA1 cells stably expressing BRCA1 showed enhanced ex- physically interacts with, and regulates, cellular tran- pression of the p53 target genes at 321C, including p21, scription factors such as p53, STAT1, and ZBRK1 mdm2, and cyclin G when compared to control cells (Ouchi et al., 1998, 2000; Zhang et al., 1998). A (Figure 1a). These genes were not induced in the absence role of BRCA1 in gene expression has been demon- of functional p53 at the nonpermissive (381C) tempera- strated by cDNA microarray analysis in which tetra- ture. We also analysed the effect of the experimental cycline-regulatable BRCA1 induced GADD45, a treatment on cell cycle distribution by flow cytometry tumor suppressor gene that is a downstream target of the p53 pathway and several other genes (Kastan et al, with propidium iodide staining. Upon shift to the permissive temperature, VhD–BRCA1 cells showed a 1992; Harkin et al., 1999). A recent report showed that dramatic reduction in the fraction of S phase cells, from BRCA1 activation of the GADD45 promoter can be mediated through the OCT-1 and CAAT motifs 49.5 to 2.9%, as well as a significant increase of the G0/G1 population (36–83%) within 2 days (Figure 1b). and physical associations of BRCA1 with transcription Although control VhD cells also showed decrease in the factor Oct-1 and NF-YA, suggesting that BRCA1 S phase population, presumably as a result of wt p53 can upregulate its target genes through protein–protein activity at 321C, it was to a lesser extent (from 52.5 to interactions (Fan et al., 2002). The potential role played 13.7%, Figure 1b). Since VhD/BRCA1 cells showed an by BRCA1 in conjunction with DNA damage response induction of the G0/G1 population without significant mechanisms is well represented by its function in increase of apoptosis, we next investigated the reversi- regulating the expression of GADD45 and is also bility of growth arrest observed in these cells upon a involved in the DNA repair pathway (Tran et al., 2002). shift from 32 to 381C. We assessed the long-term growth Of particular interest in view of these similarities between p53 and BRCA1 is the previously observed potential using a clonogenic assay. VhD-pcDNA3 or VhD/BRCA1 cells were seeded at about 100 cells per p53-dependent downregulation of BRCA1 (Arizti et al., 60 mm plate and maintained at 321C for varying time 2000; MacLachlan et al., 2000a, b). Previously, we and periods followed by a temperature shift to 381C. others showed that exogenously induced p53 causes Cultures were subsequently maintained at 381C for downregulation of BRCA1 and BRCA2 levels (Arizti another 5 days followed by fixation and Giemsa et al., 2000; MacLachlan et al., 2000a, b; Wang et al., staining. The number of colonies was counted and 2001). In addition, in response to physiological stress plotted as shown in Figure 1c. Maintenance of the cells including ionizing radiation, both BRCA1 mRNA and at 321C for 5 or more days resulted in marked reduction protein levels decrease prior to cell cycle arrest in a p53- of the ability to form colonies in both cell lines. The dependent manner (Arizti et al., 2000). Other studies also indicated that functional p53 was required for decrease in colony numbers was greater when BRCA1 and functional p53 were coexpressed (VhD/BRCA1). downregulation of BRCA1 by genotoxic stresses (Ma- These experiments demonstrated that BRCA1 is in- cLachlan et al., 2000a, b). In an attempt to further understand the functional volved in p53-mediated growth suppression rather than apoptosis. relation between the two tumor suppressor genes, we re- expressed BRCA1 in p53-activated cells and characterized the resulting phenotypes. Finally, using DNA BRCA1-mediated cell survival in response to genotoxic microarray technology, the ectopic expression of p53, stress BRCA1, or both in p53/BRCA1-null mouse embryonic fibroblasts (MEF) was investigated with specific refer- The effect of BRCA1 on p53-induced growth arrest was ence to gene expression changes. further examined using BRCA1(À/À)/p53(À/À) MEFs

Oncogene BRCA1 effects on p53 function PP Ongusaha et al 3751

Figure 1 Cooperative effects between BRCA1 and p53 on the expression of p53 target genes and p53-mediated cell cycle arrest. VhD cells expressing a ts-p53 mutant were stably transfected with a cDNA encoding human BRCA1 (VhD/BRCA1). (a) Expression of exogenous and endogenous BRCA1 and p53 target genes including p21, mdm2, and cyclin G in VhD/pcDNA3 and VhD/BRCA1 cells. RNAs were prepared from VhD/BRCA1 or control VhD cells incubated for 2 days at the indicated temperatures as described in Material and methods. (b) FACS analysis of VhD-pcDNA3 and VhD/BRCA1 cells at permissive and nonpermissive temperatures. Percentages of the cells in each phase of the cell cycle are indicated. (c) Irreversible growth inhibition of VhD/BRCA1 cells after p53 induction at 321C. The percent recoverable colonies for each condition is normalized to the number at 381C. Cultures were subsequently maintained at 381C for 5 days followed by ﬁxation and Giemsa staining. All time point studies were performed in triplicate and the means plotted using a previously published protocol (Xu et al., 1999, radiation (5 Gy). Expression levels of each adenovirus 2001). MEFs used in the study were derived from mice were examined by Western blot analysis and shown in in which both alleles of p53 and BRCA1 exon 11 had Figure 2a (left panel). Following infection of been deleted (Xu et al., 1999). BRCA1(À)/p53(À) MEFs BRCA1(À)/p53(+) MEFs with adenovirus expressing were stably infected with wt-p53-expressing retrovirus LacZ (Ad-LacZ) or BRCA1 (Ad-BRCA1) and g- (pBabe-p53) or vector (pBabe) alone to obtain radiation treatment, an increased level of p21 BRCA1(À)/p53(+) and BRCA1(À)/p53(À) MEFs. expression was observed while infection of adenoviruses These cells were infected with adenoviruses expressing without g-radiation did not affect p21 expression. LacZ or BRCA1 for 24 h and were exposed to g- Moreover, g-radiation with Ad-LacZ infection increased

Oncogene BRCA1 effects on p53 function PP Ongusaha et al 3752

Figure 2 Protective effect of BRCA1 in response to g-irradiation in the presence of p53. (a) MEFs deﬁcient for both BRCA1 and p53 were stably infected with control (pBabe) or p53 retrovirus (pBabe-p53). MEFs (2 Â 105) were further infected with adenoviruses expressing LacZ or BRCA1 with or without g-irradiation (5 Gy). The left panel shows a Western blot analysis using lysates prepared from cells infected with adenoviruses and exposed to g-radiation (5 Gy). Western blotting was performed using antibodies against p21, PUMA, p53R2, and b-actin. Cell death rates were determined by trypan blue exclusion for the different time courses. Middle panel, BRCA1(À)/p53(À) MEFs; right panel, BRCA1(À)/p53(+) MEFs. (b) Cell cycle distribution by FACS analysis of HCC1937 expressing GFP (HCC1937-IRES GFP) and HCC1937 expressing wt-BRCA1 infected Ad-p53 following g-irradiation (5 Gy). The percentages of each cell cycle phase were calculated and represented. Error bars indicate+s.d. of three independent experiments with duplicate plates

a p53-target proapoptotic protein PUMA but exogen- and not to enhance apoptosis, and this inhibition may ous BRCA1 abrogated PUMA induction in response to involve the reduction of PUMA expression level. g-radiation (Figure 2a, right panel). However, another Collaborative effect of BRCA1 on p53-induced growth proapoptotic p53-target protein p53R2 was induced by suppression was also examined in the BRCA1-mutated g-radiation in either Ad-LacZ- or Ad-BRCA1-infected HCC1937 breast cancer cell line. Vector or BRCA1- BRCA1(À)/p53(+) MEFs. Cell death rates were then introduced HCC1937 cells were infected with either determined by trypan blue exclusion in the different time Ad-LacZ or Ad-p53 24 h prior to exposing them to g- courses. BRCA1(À)/p53(À) MEFs did not show sig- irradiation (5 Gy). At 48 h following treatment with niﬁcant cell death after g-radiation, and BRCA1 g-irradiation, cells were collected and their cell cycle expression did not affect cell proliferation (Figure 2a, distribution was determined using ﬂow cytometry. As middle panel). Although BRCA1(À)/p53(+) MEFs shown in Figure 2b, coexpression of BRCA1 and p53 showed reduced cell viability and cell death 36–48 h (HCC-IRES BRCA1+p53) following exposure to g- after g-radiation, BRCA1 expression protected p53- irradiation lowered the apoptosis population (sub-G1) mediated apoptosis in response to g-radiation (Figure 2b, compared to that of cells in which only right panel). These results demonstrate that BRCA1 HCC1937+GFP (HCC-IRES GFP+p53) war, ex- collaborates with activated p53 to inhibit cell growth pressed only by p53. Although no change was observed

Oncogene BRCA1 effects on p53 function PP Ongusaha et al 3753

Figure 3 BRCA1 cooperates with wt p53 to induce the senescence phenotype. BRCA1(À/À), p53(À/À) MEFs were infected with either Ad-GFP, Ad-BRCA1, Ad-p53, or both BRCA1 and p53, and analysed 4 days postinfection. SA-b-gal staining of representative fields. The percentages of cells expressing SA-b-gal were quantified by inspecting 200 cells per 10 mm plate three times via three independent experiments in the G0/G1phase, coexpression of BRCA1 and p53 in enlarged and flat morphology, whereas Ad-BRCA1 response to g-irradiation increased G2/M population or Ad-GFP alone did not induce significant change from B62 to 72%. These results are consistent with the in morphology. Since MEFs coinfected with Ad- recent reports that BRCA1 regulates the G2/M check- BRCA1 and Ad-p53 showed characteristic morphology point after ionizing irradiation by activating key commonly observed in senescent cells, we examined effectors (Lee, 2002; Xu et al., 2001; Yarden et al., whether these MEFs expressed senescence-associated 2002). These results strongly suggest that BRCA1 is b-galactosidase (SA-b-gal), aa senescence-specific involved in the G2/M checkpoint induced by p53 and marker detected by incubating cells at pH 6.0 with allows increased survival following genotoxic stress in 5-bromo-4-chloro-3-indolye b-d-galactoside (X-gal) the presence of p53. (Dimri et al., 1995). As shown in Figure 3 (left panel), B60% of MEFs coinfected with Ad-BRCA1 BRCA1 cooperates with p53 in promoting a senescence- and Ad-p53 became positive for SA-b-gal staining like phenotypess within 4 days after infection, whereas MEFs infected with Ad-BRCA1 alone or Ad-GFP alone showed We next assessed cell morphology induced by expression no significant staining (o5%). MEFs infected of BRCA1 and p53 in BRCA1(À)/p53(À) MEFs. Cells with Ad-p53 alone (BRCA1(À)/p53(+) MEF) were infected with adenovirus-expressing green showed a slight increase in SA-b-gal activity (B28% fluorescent protein (GFP) (Ad-GFP), p53 (Ad-p53), staining). These data further confirm the finding BRCA1 (Ad-BRCA1), or both p53 and BRCA1, and that exogenous BRCA1 expression cooperates with then incubated for 3 days. MEFs infected with either p53 to promote irreversible arrest in VhD cells Ad-p53 or both Ad-BRCA1 and Ad-p53 displayed an (Figure 1c).

Oncogene BRCA1 effects on p53 function PP Ongusaha et al 3754

Figure 4 Expression levels of BRCA1, p53, and their responsive genes after infection of adenoviruses expressing LacZ, BRCA1, or p53. Western blot was performed using antibodies against BRCA1, p53, and p21. Northern blot analysis of total RNA (20 mg) following infections of the indicated adenoviruses was performed sequentially using a 32P-labeled probe against BRCA1, p53, p21, PERP, EST clone (AW 123567), and 36B4 (loading control)

Microarray analysis of p53- and BRCA1+p53-regulated enhanced a subset of p53-induced genes among the genes genes identified, which are likely to be involved in cell proliferation. These include p21Waf1 (cdk regulator, Previous studies have demonstrated that BRCA1 func- from 47-fold to 64-fold), PERP (apoptosis, from 24-fold tions as a coactivator of p53-mediated transcription, to 45.3-fold), cyclin G (mdm2 phosphorylation, from suggesting that regulation of specific p53 target genes by 14-fold to 18.4-fold), a-dgk (signaling, from 6.0-fold to BRCA1 is crucial for the p53-induced phenotype. To 9.2-fold). The above data strongly support a model in investigate the transcriptional programs that underlie which BRCA1 functions as a coactivator for p53. We the cellular response mediated by the mutual effects of also screened cDNA clones that are up- or down- p53 and BRCA1 as described in Figures 1 and 3, we regulated by Ad-BRCA1 infection of BRCA1(À)/ used cDNA microarray technology. Total RNAs were p53(À) MEFs (Table 2). Unlike p53 expression, BRCA1 isolated from BRCA1(À/À), p53(À/À) MEFs infected expression alone did not significantly affect the gene with Ad-LacZ (control), Ad-BRCA1, Ad-p53, or both expression profile in these cells although certain genes Ad-BRCA1 and Ad-p53, respectively, and fluorescent were minimally changed (approximately twofold or cDNA probes were generated and used for differential less). These results suggest that BRCA1 alone is not hybridization with a U74A murine cDNA microarray sufficient to influence transcription and that BRCA1 representing 11 000 independent genes (Affimetrix Inc.). contributes to regulation of gene expression through Expression levels of several genes induced by BRCA1 transcription factor(s) such as p53. It was previously and p53 after each adenovirus infection were confirmed reported that overexpression of BRCA1 induces the by Western and Northern blot analyses (Figure 4). Of DNA damage-responsive gene GADD45 and initiates note, coexpression of BRCA1 and p53 enhanced apoptosis in a p53-independent manner (Harkin et al., expression levels of the tested genes including p21Waf1, 1999). In our microarray experiments, Gadd45 was compared to that of p53 alone. This result is consistent upregulated only 1.4-fold in response to BRCA1 with recent reports that BRCA1 overexpression stabi- expression in BRCA1(À)/p53(À) MEFs; a recent report lizes wt p53 and then regulates transcription of growth indicates that GADD45 was induced on average 1.3- arrest genes (MacLachlan et al., 2000a, b; Welcsh et al., fold by a BRCA1-inducible system (Welcsh et al., 2002). 2002). A list of genes upregulated by expression of p53 Higher induction of GADD45 in response to BRCA1 or p53+BRCA1 is provided in Table 1, in which we might require higher levels of BRCA1 overexpression. identify known (B10%) as well as novel (B90%) p53- Genes regulated by BRCA1 art diverse in function and induced genes. Coexpression of BRCA1 and p53 play an important role in the DNA damage response,

Oncogene BRCA1 effects on p53 function PP Ongusaha et al 3755 Table 1 Comparative analysis of p53- and BRCA1+p53-regulated genes Regulation (fold change) Accession no. Function Description p53 p53+BRCA1 AW048937 Cell cycle inhibitor p21/Waf1 47 64.0 U36488 Stem cell phosphatase Esp 42 59.7 AI854029 Apoptosis PERP 24 45.3 L28819 Differentiation, aging Involucrin 24 17.1 U89491 Cancer susceptibility Eph1 21 15.0 AI851387 Unknown EST 17 17.1 L49507 Cell cycle Cyclin G 14 18.4 AW123567 Unknown EST 13 21.1 AI853375 Oncogene mdm2 9.1 11.3 L22545 ECM Procollagen type XVIII 8.5 16.0 U22262 RNA editing Apobec-1 8.0 9.9 U60020 Protein transport ABC 7.5 8.6 AB021961 Tumor suppression Mutant p53 7.5 11.3 AI846934 Adipocyte differentiation Lpin-1 6.5 8.6 AF085219 Signal transduction a-dgk 6.0 9.2 AJ131851 Tumor invasion cathepsin F 6.0 8.6 AF110520 Transmembrane a-galactosyltransferase 5.3 8.0 AI849939 Apoptosis RTP801 5.3 7.5 X04653 Alloantigen Ly-6E.1 4.9 6.1 AI846214 Development TDAG51 4.9 5.1 AF012923 Transcription Wig-1 4.9 4.9 X70296 Cell migration Serpin-4 4.6 6.5 AJ010984 Transcription matn4 4.6 7.0 AJ010108 Homeostasis AK-1 4.3 5.6 U28960 Lipid signaling Phospholipid transfer protein 6.5 D88792 Development LIM-1 4.0 5.2 Z31362 Cell migration Tx01 4.0 3.2 Y17793 Development Dutt1 4.0 4.2 M83649 Apoptosis Fas 4.0 5.6 AI553536 Unknown EST 4.0 4.9 AW122933 Signal transduction enpp2 3.7 5.3 AJ002390 Apoptosis Annexin 8 3.7 4.0 U57686 Apoptosis Sin 3.7 5.0 U82534 Apoptosis GITR3.7 4.2 U06119 Tumor invasion Cathepsin H 3.5 3.0 AI840339 Unknown EST 3.5 4.6 L10244 DNA repair SSAT 3.5 5.7 X93038 Development MAT8 3.5 4.2 U12961 Oxidative stress response NMO1 3.5 3.2 D78382 Cell growth tob 3.5 3.5 U40796 Xoroderma pigmentosum XP-G 3.5 5.0

3.5-fold or higher changes compared to control the cell cycle, apoptosis, extracellular matrix interaction, consequence of cell cycle inhibition (Arizti et al., 2000; chromatin remodeling, transcription, signal transduc- MacLachlan et al., 2000a, b; Wang et al., 2001). These tion, cell motility, and the like. results support the hypothesis that BRCA1 plays a role in the p53-directed phenotype. In this paper, we describe three major findings that further support this hypothesis; first, we show that p53-induced growth arrest Discussion becomes irreversible when BRCA1 is coexpressed, resulting in a senescence-like phenotype; second, we Several lines of evidence indicate that BRCA1 functions demonstrate that coexpression of BRCA1 and p53 in the p53 pathway: (i) BRCA1 physically binds to p53 protects cells from p53-mediated cell death, although and activates its transcriptional activity (Ouchi et al., expression of p53 alone induces apoptosis of 1998; Zhang et al., 1998; MacLachlan et al., 2002); (ii) BRCA1(À)/p53(À) MEFs after DNA damage; third, mouse embryos with defective BRCA1 expression show we establish the comparative expression profiles of p53, elevated levels of p21/WAF1 and mdm2, which are well BRCA1, and both p53 and BRCA1 using mouse cDNA characterized p53 target proteins (Gowen et al., 1996; microarray. Hakem et al., 1996: Xu et al., 1999); (iii) DNA damage- p53 is thought to inhibit growth through two activated p53 decreases BRCA1 mRNA and protein mechanisms: apoptosis and either reversible or perma- levels prior to cell cycle arrest and apoptosis, implying nent growth arrest in response to cellular stresses. The that BRCA1 modulation is p53-dependent and not a molecular processes underlying important p53-mediated

Oncogene BRCA1 effects on p53 function PP Ongusaha et al 3756 Table 2 BRCA1-responsive genes Accession no. Function Description Regulation (fold change) Upregulated genes (two-fold or higher) U68187 G protein signaling RGS2 2 AA710297 Unknown EST 3 AI849939 Apoptosis RTP801 2 L19932 Cytokine TGF-b 2 L22545 Extracellular matrix Procollagen/type XVIII 2 U63146 Retinol binding prtoein RBP 2 D00466 Apoptosis Apolipoprotein E 2 X56824 Oxidative stress Heme oxygenase 2 X03492 Keratin Keratin-complex 1 2 AW122933 Motility Autotaxin/Enpp2 2 X66473 Tumor progression MMP1 2 J03482 Chromatin/transcription Histone H2A 2 U00937 DNA repair GADD45 1.4

Downregulated genes (two-fold or less) U67160 Rho GTPase activating protein P190-B 2.5 AI847972 Membrane trafficking Vamp3 2.3 AW049254 Cisplatin-resistance associated LUC7A 2.3 X70058 Cytokine A7 2.2 AW049619 Cell cycle CDK8 2.1 X76850 MAPkinase-activated kinase 2 Mapkapk2 2.0 D83033 Nuclear protein NP220 2.0 AA727482 Differentiation Scel 2.0 U89352 Lipid signaling Lysophospholipasel 2.0 Z67747 Zinc-finger transcription factor ZT3 2.0 AF041847 Cardiac ankyrin-repeat protein MCARP 2.0 AW049275 Dual-specificity phosphatase Dusp12 2.0 AI843564 Nuclear-pore translocation TPR2.0 AA982124 Chromatin remodeling protein SNF2H 2.0 AI595322 Centromere/chromosome duplication HEC 2.0

decisions involving cell death or survival are not well evidence that BRCA1 is able to stabilize p53 in wt p53- understood. We found that MEFs derived from mice expressing cells but affects the expression of only a with deletions of exon 11 of BRCA1 that were subjected subset of known p53-regulated genes that are involved to forced expression of BRCA1 survived longer and in growth arrest and/or DNA repair rather than were less sensitive to genotoxic drugs. The survival effect apoptosis (MacLachlan et al., 2002) Forced BRCA1 mediated by BRCA1 was also described in recent expression in a p53-induced system demonstrated that reports: in the presence of BRCA1, cells did not undergo BRCA1 can enhance the levels of expression of p53- p53-mediated apoptosis (Ouchi et al., 1998; MacLachlan target genes (p21, mdm2, and cyclin G). These findings et al., 2000a, b, 2002). More recent studies indicate that suggest that the cooperative interactions between p53 BRCA1 overexpression in BRCA1-null or mutant cells and BRCA1, and subsequent p53-mediated downregu- stabilizes wt p53 but promotes survival rather than lation of BRCA1 expression, may play an important apoptosis (MacLachlan et al., 2002). These findings are role in the coordinated cellular response to DNA consistent with recent reports indicating that reintroduc- damage. Moreover, in our microarray screening, a set tion of BRCA1 into the mutant BRCA1-expressing cell of p53-induced genes including p21 was further induced line (HCC1937) allows for increased cell survival in both BRCA1 and p53-re-expressing MEFs. This following irradiation (Scully et al., 1999). These data supports the hypothesis that BRCA1 cooperates with imply that BRCA1 not only induces growth arrest in the p53 to promote cell survival in a way that may lead to presence of p53 activation but also represses apoptosis irreversible arrest. Further increase of p21 might be by shutting off the p53-specific apoptosis regulators. responsible for the senescence phenotype, since previous Conditional activation of p53 in p53-deficient cells reports indicate that p21 overexpression induced senes- induces irreversible cell cycle arrest with senescence cence in a p53-independent manner (Macip et al., 2002). features (Sugrue et al., 1997; Ferbeyre et al., 2002). Taken together, our results provide evidence that Here, we also found that reintroduction of p53 in BRCA1 can influence p53 function toward growth BRCA1- and p53-null MEFs induced senescence to a arrest or senescence, rather than toward an apoptosis lesser degree. However, reintroduction of both BRCA1 pathway. This effect may be achieved by regulating the and p53 markedly increased the frequency of the expression of a subset of p53 target genes. Identification senescence phenotype. These results would support the and characterization of these downstream genes will hypothesis that BRCA1 selectively influences p53 provide information that will be essential in under- activity in a way that may lead to the specific cellular standing the regulation of BRCA1’s effects on p53 outcome of senescence. A recent study presented strong activity.

Oncogene BRCA1 effects on p53 function PP Ongusaha et al 3757 Materials and methods g-Irradiation Cells were seeded at a cell density of 2 Â 105 in a p100 culture Cell lines and culture conditions dish with DMEM and 10% FBS. The cells were exposed to Mouse fibroblast cell line VhD was maintained in DMEM plus different doses of g-irradiation. Cells were lysed at the 10% fetal bovine serum (FBS) with the addition of 100 mg/ml indicated times. hygromycin for VhD cells as described previously (Wu et al., 1993); 750 mg/ml of geneticin was added for selection of BRCA1-expressing cell clones. VhD cells (50% confluent) were MEF survival after g-radiation transfected with pcDNA3-BRCA1 plasmid using lipofectamin g-Radiation-induced phenotypes in BRCA1-mutant MEFs 2000 (Gibco BRL). In transient cotransfection experiments, were examined using a protocol described previously (Lee the total number of plasmids was kept constant with an empty et al., 2000). Cells were infected with adenoviruses expressing vector. BRCA1-re-expressing HCC1937 cells and BRCA1(À/À)/ GFP or BRCA1 for 48 h and then treated with g-radiation p53(À/À) MEFs were maintained as described previously (5 Gy); their viability was determined using trypan blue (Scully et al., 1999; Xu et al., 1999). Stable lines expressing staining. human p53 were generated by transduction of MEFs (passage 1) with retrovirus produced by pBabepuro carrying p53 cDNA and BOSC23 packaging cells after puromycin selection (7 DNA microarray days, 1.5 mg/ml). Affymetrix GeneChips were used for hybridization. Four sets Northern blot analysis of a murine expression array (U74A murine cDNA microarray) were hybridized with fluorescently labeled rDNA Total RNA was extracted, denatured, and subjected to probes derived from total RNAs extracted from MEFs electrophoresis through a 1% agarose–formaldehyde gel infected with Ad-GFP, Ad-BRCA1, Ad-p53, or both Ad- (20 mg total RNA per lane) and transferred to a nylon BRCA1 and Ad-p53. membrane (Bio-Rad). Hybridization was performed with 32P- labeled probes prepared by the randomly primed DNA labeling method for the indicated genes. S-A-b-gal staining

Western blot analysis Cells were cultured for the indicated times, washed in PBS, and fixed with 2% formaldehyde/0.2% glutaraldehyde in PBS for Cells were washed twice with ice-cold PBS with 2 mm sodium 5 min at room temperature. The modified method for SA-b-gal vanadate and lysed in EBC lysis buffer as described previously staining of MEFs was used as previously described (Ferbeyre (Arizti et al., 2000). Lysates were cleared by centrifugation at et al., 2002). Cells were incubated in X-gal at a slightly lower 14 000 r.p.m. for 20 min at 41C. Protein concentrations were pH, 5.75, to increase the sensitivity of the assay as described then determined using a BCA protein assay kit (Pierce). earlier (Dimri et al., 1995). The percentage of cells expressing Approximately 40 mg of total cellular protein per sample was SA-b-gal was quantified by inspecting 200 cells per 10 mm subjected to SDS–Polyacrylamide gel electro phoresis (PAGE) plate three times in three independent experiments. and transferred to an Immobilon (Milipore) polyvinylidene difluoride filter. Antibodies included 421 monoclonal for p53, Ab-1 monoclonal for p21 (Oncogene Research Products), and monoclonal antibodies for BRCA1 (Ab1 and Ab2, Oncogene Abbreviations Research Products; 17F8 and 8F7, GeneTex). PUMA and FACS, Fluorescence-activated cell sorting; FBS, fetal bovine p53R2 antibodies were purchased from ProSci Inc. and serum; GFP, Green fluorescence protein; MEF, Mouse ANASPEC Inc., respectively. embryonic fibroblast; PAGE, Polyacrylamide gel electrophoresis; SA-b-gal, Senescence-associated b-galactosidase. Fluorescence-activated cell serting (FACS) analysis Cells were pelleted at 1000 r.p.m. and washed once with 10 ml of ice-cold PBS. The resultant pellets were resuspended in 1 ml of cold PBS. Ethanol, 80%, prechilled at À201C, was added Acknowledgements drop-wise with periodical vortexing to mix the cells. The We thank K-P Lu for useful comments and suggestions, M resultant mixture was kept on ice for 60 min. Cells were Ouchi for technical assistance, and M Meyer for proofreading permeabilized in 0.5% Triton X-100, 230 mg/ml RNase A, and of the manuscript. We thank RScully for HCC1937-IRES propidium iodide to 50 mg/ml in PBS. Samples were kept at BRCA1 cells, A Levine and X Wu for providing VhD cells, B 371C for 30 min followed by flow cytometry analysis (Becton Vogelstein for the adenovirus expression system and J Yu for Dickinson FACScan). Data were processed with VERITY PUMA antibodies. This work was supported in part by Grants ModFit v5.2 Microsoft Windows software for DNA distribu- CA78356, CA85214, and CA80058 from the National In- tion analysis. stitutes of Health.

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