BRCA1 shifts p53-mediated cellular outcomes towards ... - Nature

Oncogene (2003) 22, 3749–3758

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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 1 Cancer 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; 3 Department 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 enhance p53 transcription, whereas activated p53 represses BRCA1 transcription. To further understand the functional interaction of these proteins, we investigated the role of BRCA1 in p53-induced phenotypes. We found that BRCA1 when subjected to forced expression acts synergistically with wild-type p53, resulting in irreversible growth arrest, as shown by VhD mouse fibroblast cells expressing a temperature-sensitive mutant of p53. Furthermore, reintroduction of both BRCA1 and p53 into BRCA1( / )/p53( / ) mouse embryonic fibroblasts markedly increased the senescence phenotype compared to that induced by p53 alone. In particular, we found that BRCA1 expression attenuated p53-mediated cell death in response to c-irradiation. Moreover, microarray screening of 11 000 murine genes demonstrated that a set of genes upregulated by p53 is enhanced by coexpression of BRCA1 and p53, suggesting that BRCA1 and p53 exert a promoter selectivity leading to a specific phenotype. Taken together, our results provide evidence that BRCA1 is involved in p53-mediated growth suppression rather than apoptosis. Oncogene (2003) 22, 3749–3758. doi:10.1038/sj.onc.1206439 Keywords: BRCA1; P53; Senescence; Microarray; DNA damage

Introduction The p53 tumor suppressor gene, which plays a crucial role in cancer progression, is inactivated in most human tumors (Levine, 1997; Vogelstein et al., 2000). Depending on cell context, wild-type (wt) p53 limits cellular proliferation in response to DNA damage and other cellular stresses by inducing cell cycle arrest, apoptosis, *Correspondence: SW Lee, Harvard Institutes of Medicine, Room 921, 4 Blackfan Circle, Boston, MA 02115, USA; E-mail: [email protected] 5 Contributed equally to the work Received 5 November 2002; revised 27 January 2003; accepted 30 January 2003

or senescence. BRCA1 is also a tumor suppressor gene and a number of observations have implicated it in the cellular response to DNA damage (Scully et al., 1997; Cortez et al., 1999; Lee et al., 2000; Li et al., 2000; Scully and Livingston, 2000; Tibbetts et al., 2000; Wang et al., 2000). Following DNA damage, BRCA1 becomes hyperphosphorylated by ATM (Cortez et al., 1999) and hCds1/chk2 (Lee et al., 2000) and relocalizes to complexes containing PCNA (Scully et al., 1997). In addition, BRCA1 plays an important role in transcription-coupled repair and in control of cell cycle arrest following DNA damage (Abbott et al., 1999; Scully and Livingston, 2000; Wang et al., 2000). Like p53, BRCA1 also has an important role in maintaining genomic integrity (Scully and Livingston, 2000). This is strongly supported by data showing that murine embryos carrying a BRCA1 null mutation exhibit hypersensitivity to DNA damage and chromosomal abnormalities, probably because of defective G2/M checkpoint control and improper centrosome duplication (Scully et al., 1999; Somasundaram et al., 1999; MacLachlan et al., 2000a, b). BRCA1 nullizygous mice show embryonic lethality in early stage of development associated with a proliferation deficit (Gowen et al., 1996: Hakem et al., 1996; Liu et al., 1996; Xu et al., 1999, 2001). BRCA1 nullizygotes also show spontaneous abnormality of chromosome structure with an increased level of p21/WAF1 in the absence of extrinsic DNA damage. A BRCA1 exon11 knockout (Brca1D11D11) embryo died late in gestation as a result of widespread apoptosis, but elimination of one p53 allele rescued this embryonic lethality (Xu et al., 1999). Although the mice were viable, most female Brca1D11D11p53+/ mice developed mammary tumors with loss of the remaining p53 allele within 6–12 months (Xu et al., 1999). These studies also clearly showed that the p53 protein regulates BRCA1 transcription both in vitro and in vivo, and that BRCA1 participates in p53 accumulation after DNA damage through regulation of its phosphorylation and mdm2 expression (Somasundaram et al., 1997; Ouchi et al., 1998; Zhang et al., 1998; MacLachlan et al., 2000a, b). Thus, several new lines of evidence indicate that loss of BRCA1

BRCA1 effects on p53 function PP Ongusaha et al

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is not sufficient for malignant transformation but rather triggers multiple genetic alterations, including inactivation of p53 and activation of a number of oncogenes that ultimately result in mammary tumorigenesis (Brodie et al., 2001; Xu et al., 2001; Venkitaraman, 2002). A significant body of evidence implicates BRCA1 in the regulation of transcription (Chapman and Verma, 1996; Monteiro et al., 1996; Somasundaram et al., 1997; Ouchi et al., 1998). BRCA1 has an N-terminal ring finger domain and C-terminal transcription activation domain that activates transcription when fused to a DNA-binding domain (Chapman and Verma, 1996; Monteiro et al., 1996). Breast cancer-associated mutations in the BRCT domain abolish this activity, supporting the hypothesis that transcriptional regulation by BRCA1 is crucial for tumor suppression. Notably, the BRCT domain binds to several transcription activators or repressors including CBP/p300, CtIP/ CtBP, and histone deacetylases, HDAC (Deng and Brodie, 2000). Previous studies have shown that BRCA1 physically interacts with, and regulates, cellular transcription factors such as p53, STAT1, and ZBRK1 (Ouchi et al., 1998, 2000; Zhang et al., 1998). A role of BRCA1 in gene expression has been demonstrated by cDNA microarray analysis in which tetracycline-regulatable BRCA1 induced GADD45, a tumor suppressor gene that is a downstream target of the p53 pathway and several other genes (Kastan et al, 1992; Harkin et al., 1999). A recent report showed that BRCA1 activation of the GADD45 promoter can be mediated through the OCT-1 and CAAT motifs and physical associations of BRCA1 with transcription factor Oct-1 and NF-YA, suggesting that BRCA1 can upregulate its target genes through protein–protein interactions (Fan et al., 2002). The potential role played by BRCA1 in conjunction with DNA damage response mechanisms is well represented by its function in regulating the expression of GADD45 and is also involved in the DNA repair pathway (Tran et al., 2002). Of particular interest in view of these similarities between p53 and BRCA1 is the previously observed p53-dependent downregulation of BRCA1 (Arizti et al., 2000; MacLachlan et al., 2000a, b). Previously, we and others showed that exogenously induced p53 causes downregulation of BRCA1 and BRCA2 levels (Arizti et al., 2000; MacLachlan et al., 2000a, b; Wang et al., 2001). In addition, in response to physiological stress including ionizing radiation, both BRCA1 mRNA and protein levels decrease prior to cell cycle arrest in a p53dependent manner (Arizti et al., 2000). Other studies also indicated that functional p53 was required for downregulation of BRCA1 by genotoxic stresses (MacLachlan et al., 2000a, b). In an attempt to further understand the functional relation between the two tumor suppressor genes, we reexpressed BRCA1 in p53-activated cells and characterized the resulting phenotypes. Finally, using DNA microarray technology, the ectopic expression of p53, BRCA1, or both in p53/BRCA1-null mouse embryonic fibroblasts (MEF) was investigated with specific reference to gene expression changes. Oncogene

Results Collaborative effects between BRCA1 and p53 on expression of p53-target genes and growth arrest To explore the biological significance of BRCA1 repression by p53, we first generated stable BRCA1 transfectants in VhD cells, which contain p53 in a temperature-sensitive form (p53val135) (Wu et al., 1993) and in p53-null fibroblasts of the 10.1 line, and then determined proliferation at nonpermissive (381C) and permissive (321C) temperatures for p53 activity. VhD cells were transfected with a BRCA1 expression plasmid (pcDNA3-BRCA1) or vector alone (pcDNA3) and selected with G418 at 381C to avoid p53 activation. Consistent with previous results, a temperature shift to 321C in control cells (VhD-pcDNA3) resulted in a significant reduction of endogenous BRCA1 mRNA expression while known p53-target genes were induced following p53 activation (Figure 1a). However, VhD cells stably expressing BRCA1 showed enhanced expression of the p53 target genes at 321C, including p21, mdm2, and cyclin G when compared to control cells (Figure 1a). These genes were not induced in the absence of functional p53 at the nonpermissive (381C) temperature. We also analysed the effect of the experimental treatment on cell cycle distribution by flow cytometry with propidium iodide staining. Upon shift to the permissive temperature, VhD–BRCA1 cells showed a dramatic reduction in the fraction of S phase cells, from 49.5 to 2.9%, as well as a significant increase of the G0/G1 population (36–83%) within 2 days (Figure 1b). Although control VhD cells also showed decrease in the S phase population, presumably as a result of wt p53 activity at 321C, it was to a lesser extent (from 52.5 to 13.7%, Figure 1b). Since VhD/BRCA1 cells showed an induction of the G0/G1 population without significant increase of apoptosis, we next investigated the reversibility of growth arrest observed in these cells upon a shift from 32 to 381C. We assessed the long-term growth potential using a clonogenic assay. VhD-pcDNA3 or VhD/BRCA1 cells were seeded at about 100 cells per 60 mm plate and maintained at 321C for varying time periods followed by a temperature shift to 381C. Cultures were subsequently maintained at 381C for another 5 days followed by fixation and Giemsa staining. The number of colonies was counted and plotted as shown in Figure 1c. Maintenance of the cells at 321C for 5 or more days resulted in marked reduction of the ability to form colonies in both cell lines. The decrease in colony numbers was greater when BRCA1 and functional p53 were coexpressed (VhD/BRCA1). These experiments demonstrated that BRCA1 is involved in p53-mediated growth suppression rather than apoptosis. BRCA1-mediated cell survival in response to genotoxic stress The effect of BRCA1 on p53-induced growth arrest was further examined using BRCA1( / )/p53( / ) MEFs


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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, 2001). MEFs used in the study were derived from mice in which both alleles of p53 and BRCA1 exon 11 had been deleted (Xu et al., 1999). BRCA1( )/p53( ) MEFs were stably infected with wt-p53-expressing retrovirus (pBabe-p53) or vector (pBabe) alone to obtain BRCA1( )/p53(+) and BRCA1( )/p53( ) MEFs. These cells were infected with adenoviruses expressing LacZ or BRCA1 for 24 h and were exposed to g-

radiation (5 Gy). Expression levels of each adenovirus were examined by Western blot analysis and shown in Figure 2a (left panel). Following infection of BRCA1( )/p53(+) MEFs with adenovirus expressing LacZ (Ad-LacZ) or BRCA1 (Ad-BRCA1) and gradiation treatment, an increased level of p21 expression was observed while infection of adenoviruses without g-radiation did not affect p21 expression. Moreover, g-radiation with Ad-LacZ infection increased Oncogene


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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 (2 105) 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( )/p53( ) MEFs; right panel, BRCA1( )/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 exogenous BRCA1 abrogated PUMA induction in response to g-radiation (Figure 2a, right panel). However, another proapoptotic p53-target protein p53R2 was induced by g-radiation in either Ad-LacZ- or Ad-BRCA1-infected BRCA1( )/p53(+) MEFs. Cell death rates were then determined by trypan blue exclusion in the different time courses. BRCA1( )/p53( ) MEFs did not show significant cell death after g-radiation, and BRCA1 expression did not affect cell proliferation (Figure 2a, middle panel). Although BRCA1( )/p53(+) MEFs showed reduced cell viability and cell death 36–48 h after g-radiation, BRCA1 expression protected p53mediated apoptosis in response to g-radiation (Figure 2b, right panel). These results demonstrate that BRCA1 collaborates with activated p53 to inhibit cell growth Oncogene

and not to enhance apoptosis, and this inhibition may involve the reduction of PUMA expression level. Collaborative effect of BRCA1 on p53-induced growth suppression was also examined in the BRCA1-mutated HCC1937 breast cancer cell line. Vector or BRCA1introduced HCC1937 cells were infected with either Ad-LacZ or Ad-p53 24 h prior to exposing them to girradiation (5 Gy). At 48 h following treatment with g-irradiation, cells were collected and their cell cycle distribution was determined using flow cytometry. As shown in Figure 2b, coexpression of BRCA1 and p53 (HCC-IRES BRCA1+p53) following exposure to girradiation lowered the apoptosis population (sub-G1) compared to that of cells in which only HCC1937+GFP (HCC-IRES GFP+p53) war, expressed only by p53. Although no change was observed


3753

Figure 3 BRCA1 cooperates with wt p53 to induce the senescence phenotype. BRCA1( / ), p53( / ) 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 response to g-irradiation increased G2/M population from B62 to 72%. These results are consistent with the recent reports that BRCA1 regulates the G2/M checkpoint after ionizing irradiation by activating key effectors (Lee, 2002; Xu et al., 2001; Yarden et al., 2002). These results strongly suggest that BRCA1 is involved in the G2/M checkpoint induced by p53 and allows increased survival following genotoxic stress in the presence of p53. BRCA1 cooperates with p53 in promoting a senescencelike phenotypess We next assessed cell morphology induced by expression of BRCA1 and p53 in BRCA1( )/p53( ) MEFs. Cells were infected with adenovirus-expressing green fluorescent protein (GFP) (Ad-GFP), p53 (Ad-p53), BRCA1 (Ad-BRCA1), or both p53 and BRCA1, and then incubated for 3 days. MEFs infected with either Ad-p53 or both Ad-BRCA1 and Ad-p53 displayed an

enlarged and flat morphology, whereas Ad-BRCA1 or Ad-GFP alone did not induce significant change in morphology. Since MEFs coinfected with AdBRCA1 and Ad-p53 showed characteristic morphology commonly observed in senescent cells, we examined whether these MEFs expressed senescence-associated b-galactosidase (SA-b-gal), aa senescence-specific marker detected by incubating cells at pH 6.0 with 5-bromo-4-chloro-3-indolye b-d-galactoside (X-gal) (Dimri et al., 1995). As shown in Figure 3 (left panel), B60% of MEFs coinfected with Ad-BRCA1 and Ad-p53 became positive for SA-b-gal staining within 4 days after infection, whereas MEFs infected with Ad-BRCA1 alone or Ad-GFP alone showed no significant staining (o5%). MEFs infected with Ad-p53 alone (BRCA1( )/p53(+) MEF) showed a slight increase in SA-b-gal activity (B28% staining). These data further confirm the finding that exogenous BRCA1 expression cooperates with p53 to promote irreversible arrest in VhD cells (Figure 1c). Oncogene


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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 genes Previous studies have demonstrated that BRCA1 functions as a coactivator of p53-mediated transcription, suggesting that regulation of specific p53 target genes by BRCA1 is crucial for the p53-induced phenotype. To investigate the transcriptional programs that underlie the cellular response mediated by the mutual effects of p53 and BRCA1 as described in Figures 1 and 3, we used cDNA microarray technology. Total RNAs were isolated from BRCA1( / ), p53( / ) MEFs infected with Ad-LacZ (control), Ad-BRCA1, Ad-p53, or both Ad-BRCA1 and Ad-p53, respectively, and fluorescent cDNA probes were generated and used for differential hybridization with a U74A murine cDNA microarray representing 11 000 independent genes (Affimetrix Inc.). Expression levels of several genes induced by BRCA1 and p53 after each adenovirus infection were confirmed by Western and Northern blot analyses (Figure 4). Of note, coexpression of BRCA1 and p53 enhanced expression levels of the tested genes including p21Waf1, compared to that of p53 alone. This result is consistent with recent reports that BRCA1 overexpression stabilizes wt p53 and then regulates transcription of growth arrest genes (MacLachlan et al., 2000a, b; Welcsh et al., 2002). A list of genes upregulated by expression of p53 or p53+BRCA1 is provided in Table 1, in which we identify known (B10%) as well as novel (B90%) p53induced genes. Coexpression of BRCA1 and p53 Oncogene

enhanced a subset of p53-induced genes among the genes identified, which are likely to be involved in cell proliferation. These include p21Waf1 (cdk regulator, from 47-fold to 64-fold), PERP (apoptosis, from 24-fold to 45.3-fold), cyclin G (mdm2 phosphorylation, from 14-fold to 18.4-fold), a-dgk (signaling, from 6.0-fold to 9.2-fold). The above data strongly support a model in which BRCA1 functions as a coactivator for p53. We also screened cDNA clones that are up- or downregulated by Ad-BRCA1 infection of BRCA1( )/ p53( ) MEFs (Table 2). Unlike p53 expression, BRCA1 expression alone did not significantly affect the gene expression profile in these cells although certain genes were minimally changed (approximately twofold or less). These results suggest that BRCA1 alone is not sufficient to influence transcription and that BRCA1 contributes to regulation of gene expression through transcription factor(s) such as p53. It was previously reported that overexpression of BRCA1 induces the DNA damage-responsive gene GADD45 and initiates apoptosis in a p53-independent manner (Harkin et al., 1999). In our microarray experiments, Gadd45 was upregulated only 1.4-fold in response to BRCA1 expression in BRCA1( )/p53( ) MEFs; a recent report indicates that GADD45 was induced on average 1.3fold by a BRCA1-inducible system (Welcsh et al., 2002). Higher induction of GADD45 in response to BRCA1 might require higher levels of BRCA1 overexpression. Genes regulated by BRCA1 art diverse in function and play an important role in the DNA damage response,


3755 Table 1 Comparative analysis of p53- and BRCA1+p53-regulated genes Regulation (fold change) Accession no.

Function

Description

p53

p53+BRCA1

AW048937 U36488 AI854029 L28819 U89491 AI851387 L49507 AW123567 AI853375 L22545 U22262 U60020 AB021961 AI846934 AF085219 AJ131851 AF110520 AI849939 X04653 AI846214 AF012923 X70296 AJ010984 AJ010108 U28960 D88792 Z31362 Y17793 M83649 AI553536 AW122933 AJ002390 U57686 U82534 U06119 AI840339 L10244 X93038 U12961 D78382 U40796

Cell cycle inhibitor Stem cell phosphatase Apoptosis Differentiation, aging Cancer susceptibility Unknown Cell cycle Unknown Oncogene ECM RNA editing Protein transport Tumor suppression Adipocyte differentiation Signal transduction Tumor invasion Transmembrane Apoptosis Alloantigen Development Transcription Cell migration Transcription Homeostasis Lipid signaling Development Cell migration Development Apoptosis Unknown Signal transduction Apoptosis Apoptosis Apoptosis Tumor invasion Unknown DNA repair Development Oxidative stress response Cell growth Xoroderma pigmentosum

p21/Waf1 Esp PERP Involucrin Eph1 EST Cyclin G EST mdm2 Procollagen type XVIII Apobec-1 ABC Mutant p53 Lpin-1 a-dgk cathepsin F a-galactosyltransferase RTP801 Ly-6E.1 TDAG51 Wig-1 Serpin-4 matn4 AK-1 Phospholipid transfer protein LIM-1 Tx01 Dutt1 Fas EST enpp2 Annexin 8 Sin GITR Cathepsin H EST SSAT MAT8 NMO1 tob XP-G

47 42 24 24 21 17 14 13 9.1 8.5 8.0 7.5 7.5 6.5 6.0 6.0 5.3 5.3 4.9 4.9 4.9 4.6 4.6 4.3

64.0 59.7 45.3 17.1 15.0 17.1 18.4 21.1 11.3 16.0 9.9 8.6 11.3 8.6 9.2 8.6 8.0 7.5 6.1 5.1 4.9 6.5 7.0 5.6 6.5 5.2 3.2 4.2 5.6 4.9 5.3 4.0 5.0 4.2 3.0 4.6 5.7 4.2 3.2 3.5 5.0

4.0 4.0 4.0 4.0 4.0 3.7 3.7 3.7 3.7 3.5 3.5 3.5 3.5 3.5 3.5 3.5

3.5-fold or higher changes compared to control

the cell cycle, apoptosis, extracellular matrix interaction, chromatin remodeling, transcription, signal transduction, cell motility, and the like.

Discussion Several lines of evidence indicate that BRCA1 functions in the p53 pathway: (i) BRCA1 physically binds to p53 and activates its transcriptional activity (Ouchi et al., 1998; Zhang et al., 1998; MacLachlan et al., 2002); (ii) mouse embryos with defective BRCA1 expression show elevated levels of p21/WAF1 and mdm2, which are well characterized p53 target proteins (Gowen et al., 1996; Hakem et al., 1996: Xu et al., 1999); (iii) DNA damageactivated p53 decreases BRCA1 mRNA and protein levels prior to cell cycle arrest and apoptosis, implying that BRCA1 modulation is p53-dependent and not a

consequence of cell cycle inhibition (Arizti et al., 2000; MacLachlan et al., 2000a, b; Wang et al., 2001). These 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 becomes irreversible when BRCA1 is coexpressed, resulting in a senescence-like phenotype; second, we demonstrate that coexpression of BRCA1 and p53 protects cells from p53-mediated cell death, although expression of p53 alone induces apoptosis of BRCA1( )/p53( ) MEFs after DNA damage; third, we establish the comparative expression profiles of p53, BRCA1, and both p53 and BRCA1 using mouse cDNA microarray. p53 is thought to inhibit growth through two mechanisms: apoptosis and either reversible or permanent growth arrest in response to cellular stresses. The molecular processes underlying important p53-mediated Oncogene


3756 Table 2 BRCA1-responsive genes Accession no.

Function

Description

Regulation (fold change)

Upregulated genes (two-fold or higher) U68187 G protein signaling AA710297 Unknown AI849939 Apoptosis L19932 Cytokine L22545 Extracellular matrix U63146 Retinol binding prtoein D00466 Apoptosis X56824 Oxidative stress X03492 Keratin AW122933 Motility X66473 Tumor progression J03482 Chromatin/transcription U00937 DNA repair

RGS2 EST RTP801 TGF-b Procollagen/type XVIII RBP Apolipoprotein E Heme oxygenase Keratin-complex 1 Autotaxin/Enpp2 MMP1 Histone H2A GADD45

2 3 2 2 2 2 2 2 2 2 2 2 1.4

Downregulated genes (two-fold or less) U67160 Rho GTPase activating protein AI847972 Membrane trafficking AW049254 Cisplatin-resistance associated X70058 Cytokine AW049619 Cell cycle X76850 MAPkinase-activated kinase 2 D83033 Nuclear protein AA727482 Differentiation U89352 Lipid signaling Z67747 Zinc-finger transcription factor AF041847 Cardiac ankyrin-repeat protein AW049275 Dual-specificity phosphatase AI843564 Nuclear-pore translocation AA982124 Chromatin remodeling protein AI595322 Centromere/chromosome duplication

P190-B Vamp3 LUC7A A7 CDK8 Mapkapk2 NP220 Scel Lysophospholipasel ZT3 MCARP Dusp12 TPR SNF2H HEC

2.5 2.3 2.3 2.2 2.1 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0

decisions involving cell death or survival are not well understood. We found that MEFs derived from mice with deletions of exon 11 of BRCA1 that were subjected to forced expression of BRCA1 survived longer and were less sensitive to genotoxic drugs. The survival effect mediated by BRCA1 was also described in recent reports: in the presence of BRCA1, cells did not undergo p53-mediated apoptosis (Ouchi et al., 1998; MacLachlan et al., 2000a, b, 2002). More recent studies indicate that BRCA1 overexpression in BRCA1-null or mutant cells stabilizes wt p53 but promotes survival rather than apoptosis (MacLachlan et al., 2002). These findings are consistent with recent reports indicating that reintroduction of BRCA1 into the mutant BRCA1-expressing cell line (HCC1937) allows for increased cell survival following irradiation (Scully et al., 1999). These data imply that BRCA1 not only induces growth arrest in the presence of p53 activation but also represses apoptosis by shutting off the p53-specific apoptosis regulators. Conditional activation of p53 in p53-deficient cells induces irreversible cell cycle arrest with senescence features (Sugrue et al., 1997; Ferbeyre et al., 2002). Here, we also found that reintroduction of p53 in BRCA1- and p53-null MEFs induced senescence to a lesser degree. However, reintroduction of both BRCA1 and p53 markedly increased the frequency of the senescence phenotype. These results would support the hypothesis that BRCA1 selectively influences p53 activity in a way that may lead to the specific cellular outcome of senescence. A recent study presented strong Oncogene

evidence that BRCA1 is able to stabilize p53 in wt p53expressing cells but affects the expression of only a subset of known p53-regulated genes that are involved in growth arrest and/or DNA repair rather than apoptosis (MacLachlan et al., 2002) Forced BRCA1 expression in a p53-induced system demonstrated that BRCA1 can enhance the levels of expression of p53target genes (p21, mdm2, and cyclin G). These findings suggest that the cooperative interactions between p53 and BRCA1, and subsequent p53-mediated downregulation of BRCA1 expression, may play an important role in the coordinated cellular response to DNA damage. Moreover, in our microarray screening, a set of p53-induced genes including p21 was further induced in both BRCA1 and p53-re-expressing MEFs. This supports the hypothesis that BRCA1 cooperates with p53 to promote cell survival in a way that may lead to irreversible arrest. Further increase of p21 might be responsible for the senescence phenotype, since previous reports indicate that p21 overexpression induced senescence in a p53-independent manner (Macip et al., 2002). Taken together, our results provide evidence that BRCA1 can influence p53 function toward growth arrest or senescence, rather than toward an apoptosis pathway. This effect may be achieved by regulating the expression of a subset of p53 target genes. Identification and characterization of these downstream genes will provide information that will be essential in understanding the regulation of BRCA1’s effects on p53 activity.


3757 Materials and methods Cell lines and culture conditions Mouse fibroblast cell line VhD was maintained in DMEM plus 10% fetal bovine serum (FBS) with the addition of 100 mg/ml 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 transfected with pcDNA3-BRCA1 plasmid using lipofectamin 2000 (Gibco BRL). In transient cotransfection experiments, the total number of plasmids was kept constant with an empty vector. BRCA1-re-expressing HCC1937 cells and BRCA1( / )/ p53( / ) MEFs were maintained as described previously (Scully et al., 1999; Xu et al., 1999). Stable lines expressing 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 days, 1.5 mg/ml). Northern blot analysis Total RNA was extracted, denatured, and subjected to electrophoresis through a 1% agarose–formaldehyde gel (20 mg total RNA per lane) and transferred to a nylon membrane (Bio-Rad). Hybridization was performed with 32Plabeled probes prepared by the randomly primed DNA labeling method for the indicated genes. Western blot analysis Cells were washed twice with ice-cold PBS with 2 mm sodium vanadate and lysed in EBC lysis buffer as described previously (Arizti et al., 2000). Lysates were cleared by centrifugation at 14 000 r.p.m. for 20 min at 41C. Protein concentrations were then determined using a BCA protein assay kit (Pierce). Approximately 40 mg of total cellular protein per sample was subjected to SDS–Polyacrylamide gel electro phoresis (PAGE) 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 Research Products; 17F8 and 8F7, GeneTex). PUMA and p53R2 antibodies were purchased from ProSci Inc. and ANASPEC Inc., respectively. 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 201C, was added drop-wise with periodical vortexing to mix the cells. The resultant mixture was kept on ice for 60 min. Cells were permeabilized in 0.5% Triton X-100, 230 mg/ml RNase A, and propidium iodide to 50 mg/ml in PBS. Samples were kept at 371C for 30 min followed by flow cytometry analysis (Becton Dickinson FACScan). Data were processed with VERITY ModFit v5.2 Microsoft Windows software for DNA distribution analysis.

g-Irradiation Cells were seeded at a cell density of 2 105 in a p100 culture dish with DMEM and 10% FBS. The cells were exposed to different doses of g-irradiation. Cells were lysed at the indicated times. MEF survival after g-radiation g-Radiation-induced phenotypes in BRCA1-mutant MEFs were examined using a protocol described previously (Lee et al., 2000). Cells were infected with adenoviruses expressing GFP or BRCA1 for 48 h and then treated with g-radiation (5 Gy); their viability was determined using trypan blue staining. DNA microarray Affymetrix GeneChips were used for hybridization. Four sets of a murine expression array (U74A murine cDNA microarray) were hybridized with fluorescently labeled rDNA probes derived from total RNAs extracted from MEFs infected with Ad-GFP, Ad-BRCA1, Ad-p53, or both AdBRCA1 and Ad-p53. S-A-b-gal staining Cells were cultured for the indicated times, washed in PBS, and fixed with 2% formaldehyde/0.2% glutaraldehyde in PBS for 5 min at room temperature. The modified method for SA-b-gal staining of MEFs was used as previously described (Ferbeyre et al., 2002). Cells were incubated in X-gal at a slightly lower pH, 5.75, to increase the sensitivity of the assay as described earlier (Dimri et al., 1995). The percentage of cells expressing SA-b-gal was quantified by inspecting 200 cells per 10 mm plate three times in three independent experiments.

Abbreviations FACS, Fluorescence-activated cell sorting; FBS, fetal bovine serum; GFP, Green fluorescence protein; MEF, Mouse embryonic fibroblast; PAGE, Polyacrylamide gel electrophoresis; SA-b-gal, Senescence-associated b-galactosidase.

Acknowledgements We thank K-P Lu for useful comments and suggestions, M Ouchi for technical assistance, and M Meyer for proofreading of the manuscript. We thank R Scully for HCC1937-IRES BRCA1 cells, A Levine and X Wu for providing VhD cells, B Vogelstein for the adenovirus expression system and J Yu for PUMA antibodies. This work was supported in part by Grants CA78356, CA85214, and CA80058 from the National Institutes of Health.

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