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RESEARCH PAPER
1 Advanced Medical Discovery Institute, Ontario Cancer Institute, Toronto, Ontario M5G 2C1, Canada; 2 Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5S 1A8, Canada; 3 The Sakaguchi Laboratory of Developmental Biology, School of Medicine, Keio University, Shinjuku, Tokyo 160-8582, Japan; 4 Department of Physiology, Faculty of Medicine, National University of Singapore, 117597 Singapore
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[Keywords: Breast cancer; T cell; genomic instability; apoptosis; cell cycle; proliferation]
Received February 10, 2004; revised version accepted April 2, 2004.
; Welcsh et al. 2000; Venkitaraman 2002). Despite its role in maintenance of genome integrity (Kinzler and Vogelstein 1997), transcriptional regulation (Scully et al. 1997a; Somasundaram et al. 1997), and chromatin remodeling (Yarden and Brody 1999; Bochar et al. 2000; Pao et al. 2000; Wang et al. 2000; Welcsh et al. 2000), the exact mechanism of tumor suppression by BRCA1 remains to be defined. BRCA1 associates with proteins that function in DNA replication and repair (Scully et al. 1997b; Wang et al. 2000), transcriptional activation (Chapman and Verma 1996; Monteiro et al. 1996; Scully et al. 1997a; Li et al. 2000), and the DNA damage response (Scully et al. 1997b; Zhong et al. 1999; Li et al. 2000; Wang et al. 2000; Welcsh et al. 2000). Cells with mutant Brca1 display defects in survival and proliferation (Gowen et al. 1996; Hakem et al. 1996; Ludwig et al. 1997; Shen et al. 1998), radiosensitivity (Shen et al. 1998; Welcsh et al. 2000), chromosomal abnormalities (Xu et al. 1999; Mak et al. 2000; Welcsh et al. 2000), p53 activation (Hakem et al. 1996), G2/M checkpoint loss (Larson et al. 1997; Xu et al. 1999), and impaired homologous recombination repair (Moynahan et al. 1999). Accordingly, the pleiotropic effects of BRCA1 mutation could be attributed to its involvement in DNA repair and transcriptional regulation.
Brca1-targeted disruption in mice results in embryonic lethality (Gowen et al. 1996; Hakem et al. 1996; Liu et al. 1996; Ludwig et al. 1997). To circumvent this limitation, conditional targeting strategies have been employed that facilitate the study of the consequences of Brca1 disruption in vivo and ex vivo (Xu et al. 1999; Mak et al. 2000). Mice that are tBrca1–/– (Mak et al. 2000) carry a targeted null mutation of Brca1 restricted to the T-cell lineage, thus bypassing the associated lethality of Brca1 germline disruption. Brca1 disruption in the T-cell compartment results in a drastic depletion of thymocytes and peripheral T cells, the accumulation of chromosomal abnormalities, and activation of p53. Thymocyte development of tBrca1–/– mice is restored in the absence of p53 or the presence of overexpressed anti-apoptotic protein Bcl2 that antagonizes the p53 pathway. These findings suggest that genomic instability and p53 activation due to the ablation of Brca1 contribute to the observed defects in T-cell development, survival, and proliferation.
Although several studies have linked the compromised viability and genomic instability of Brca1-deficient cells to the activation of p53 by genome surveillance (Hakem et al. 1997; Shen et al. 1998; Mak et al. 2000; Xu et al. 2001; Cao et al. 2003), the molecular components that comprise this response are unclear. Recent studies of p53 have elucidated upstream signaling cascades that modulate its half-life, intracellular localization, and functional activity (Giaccia and Kastan 1998; Caspari 2000; Vousden and Lu 2002). DNA damage alters the phosphorylation status of p53 and other DNA damage response proteins by triggering molecular signaling cascades that include ATM, ATR, DNA-PK, Chk1, and Chk2 kinases (for review, see Rich et al. 2000; Zhou and Elledge 2000; Nyberg et al. 2002; Shiloh 2003). A component of these genome surveillance pathways, Chk2 (also known as Cds1, for review, see Bartek et al. 2001; McGowan 2002) has been identified as a tumor suppressor, as mutations in Chk2 have been implicated in familial cancer syndromes including Li-Fraumeni (Bell et al. 1999) and low penetrance breast cancer susceptibility (Meijers-Heijboer et al. 2002; Vahteristo et al. 2002). Several studies have elucidated an ATM-Chk2-p53 DNA damage signaling cascade that is activated following DNA damage (Hirao et al. 2000; Bartek et al. 2001). As Chk2 functions in a DNA damage response pathway that result in p53 activation, we sought to determine the contribution of this kinase to phenotypes associated with Brca1 deficiency. We demonstrate that deficiency in Chk2 partially mimics the loss of p53 and rescues the defective development, growth, and cellular demise of Brca1-deficient T cells at the expense of genomic instability and increased tumorigenicity. We have also identified an important role for Chk2 in suppressing Brca1-associated breast cancer.
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) in the T-cell lineage (LckCre Brca1fl/fl "tBrca1–/–") or mammary epithelium (WapCre Brca1fl/fl "mBrca1–/–") and Chk2–/– mice (Hirao et al. 2002) were intercrossed to obtain mice deficient in both Brca1 and Chk2 in the T-cell lineage (tBrca1–/–Chk2–/–) and mammary epithelium (mBrca1–/–Chk2–/–). Thymocyte development in tBrca1–/– mice (Mak et al. 2000) is compromised and the total number of T cells is markedly reduced (Fig. 1; Supplementary Table 1). Chk2 deficiency does not markedly impact total thymus cellularity (Hirao et al. 2000, 2002), although a slight increase in the absolute numbers of CD4–CD8– population was detected (Fig. 1; Supplementary Table 1). The defective cellularity of the CD4+CD8+, CD4+, and CD8+ subsets in Brca1-deficient thymocytes was found to be Chk2-dependent, as tBrca1–/–Chk2–/– mice show complete restoration of all thymocyte and peripheral T-cell populations (Fig. 1; Supplementary Table 1).
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-irradiation ex vivo (Mak et al. 2000). This radiosensitivity is p53 dependent, as it is rescued in a p53 null background. Chk2–/– thymocytes exhibit radioresistance compared with wild-type thymocytes (Hirao et al. 2000, 2002; Takai et al. 2002). The radiosensitivity of tBrca1–/– thymocytes was also found to be dependent on Chk2, as tBrca1–/–Chk2–/– thymocytes and Chk2–/– thymocytes show equivalent resistance when assessed 8 h or 24 h after -irradiation (Fig. 2A,B). The proliferation impediment exhibited by tBrca1–/– peripheral T cells following activation with anti-CD3
+ IL-2 or anti-CD3 + anti-CD28 was attenuated in the absence of Chk2 (Fig. 2C,D). The G1 + G2/M-phase arrest in anti-CD3 + IL-2 activated tBrca1–/– cells was reversed in the absence of Chk2, suggesting that the reversal of the proliferation impediment was at least partially the result of attenuation of the cell cycle arrest (Fig. 2E).
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; Shieh et al. 2000; Hirao et al. 2002; Takai et al. 2002). Increased steady-state levels of p53 and its downstream transcriptional targets p21 and bax were observed in Brca1-deficient thymocytes (Fig. 2F). The absence of Chk2 was found to attenuate the steady-state levels of p53, p21, and bax in thymocytes deficient in Brca1, indicating the involvement of Chk2 in mediating the effect of Brca1 deficiency on p53. Increased phosphorylation levels of Chk2 were observed in Brca1 deficient thymocytes, indicative of a role for Chk2 activity in the increased steady-state level of p53 in Brca1-deficient thymocytes (Fig. 2G). Irradiation-dependent phosphorylation of Chk2 was not affected by Brca1 mutation in thymocytes (Fig. 2G). These findings indicate that Chk2 inactivation facilitates the survival and proliferation of Brca1-deficient T cells by attenuating p53-dependent apoptosis and cell cycle arrest.
Brca1 deficient thymocytes exhibit increased levels of spontaneous genomic instability (Mak et al. 2000). As genomic instability normally leads to stabilization and activation of p53, the attenuation of p53 activation in tBrca1–/–Chk2–/– T cells could be the result of a reversal in the genomic instability phenotype inherent in Brca1-deficient cells. However, the frequency of chromosomal breaks, fusions, tri-radials, and aneuploidy was markedly increased in tBrca1–/–Chk2–/– peripheral T cells compared with Chk2–/– cells (Fig. 3; Table 1), indicative of the impaired DNA damage response in the absence of Chk2. Interestingly, the spectrum of chromosomal abnormalities was consistent with those obtained from tBrca1–/–p53–/–-activated T cells; however, the frequency of all observed chromosomal abnormalities was lower (Table 1). Thus, loss of Chk2 reverses apoptosis and cell-cycle defects but not the genomic instability associated with Brca1 mutation in T cells.
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; Xu et al. 1999, 2001; Ludwig et al. 2001). In the thymus, Brca1 and p53 mutations were found to potentiate the formation of thymic lymphomas. Complete penetrance (100%) and decreased latency of tumor development was observed in tBrca1–/– p53–/– mice (p < 0.0001), and the majority of these were moribund or died by 3 mo of age (Fig. 4A,E) compared with tBrca1+/–p53–/– and p53–/– mice, which develop lymphomas or other tumors after 
6 mo of age, as anticipated from previous studies (Donehower et al. 1992). The rescue of thymocyte development and abrogation of p53 checkpoint-dependent responses in tBrca1–/–p53–/– and tBrca1–/–Chk2–/– would predict a similar cooperativity in tumorigenesis in the concomitant absence of Brca1 and Chk2. Although only 12% of tBrca1–/– mice (4/33; Fig. 4B–D) and no Chk2–/– (0/20) succumbed to tumors after 400 d of observation, 32% of tBrca1–/– Chk2–/– mice (7/22) succumbed to thymic lymphomas at an earlier onset (by 180 d, p = 0.0155; Fig. 4B,F). tBrca1–/– Chk2–/– lymphomas (and tBrca1–/–p53–/– lymphomas) were observed to be restricted to the thymus and were not observed to invade other organs such as the spleen and liver. The small proportion of tumor-burdened tBrca1–/– mice did not develop thymic lymphomas, but instead exhibited lymphomas in peripheral lymphoid compartments that invaded the liver, lungs, and kidney. The incomplete penetrance in the tumor-prone phenotype suggests that some of the p53 tumor suppressor functions are retained in the absence of Chk2.
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Mammary tumors from either mBrca1–/–Chk2–/– or mBrca1–/–p53+/– females exhibited histological features characteristic of acinar adenocarcinomas (2/4 from mBrca1–/–Chk2–/– mice and 4/4 from mBrca1–/–p53+/– mice) of varying differentiation status, with tumor cells typically organized in small glandular structures containing a central lumen and occasional luminal secretions (Fig. 5A,B,D–H). Two of the four mammary carcinomas obtained from mBrca1–/–Chk2–/– mice also displayed regions of squamous differentiation, containing keratin pearls (Fig. 5C). Local infiltration into the adjacent soft tissue without metastatic invasion into the lungs or the lymphatic system was observed.
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; Shen et al. 1998; Mak et al. 2000; Xu et al. 2001; Cao et al. 2003). The targeted disruption of Brca1 is often incompatible with cell viability, a finding that would appear incompatible with a role in tumor suppression (Venkitaraman 2002). However, the spontaneous chromosomal instability in Brca1-deficient cells might facilitate neoplastic transformation in cells with compromised responses to DNA damaging signaling through p53. Tumorigenesis in the absence of BRCA1 is facilitated by p53 deficiency; however, not all tumors from BRCA1 mutation carriers contain mutant p53, indicative of the existence of alternative pathways that facilitate transformation (Crook et al. 1997; Greenblatt et al. 2001).
Our findings indicate the loss of p53-dependent responses mediated by Chk2 may be critical for survival, development, and tumor progression triggered by loss of Brca1 function. Loss of Chk2 does not rescue the phenotype of the Brca1-deficient T cells to the same extent as loss of p53. This indicates that Chk2 mediates only a subset of p53 function. Chk2 is a component of the network of genome-surveillance pathways that coordinate cell-cycle progression with DNA repair and cell survival or death (Bartek et al. 2001; McGowan 2002). Chk2–/– cells are radioresistant and show defects in -irradiation-induced apoptosis (Hirao et al. 2000, 2002; Takai et al. 2002). The mechanism whereby Chk2 facilitates radiation-induced apoptosis is poorly understood, but is thought to involve phosphorylation and stabilization of p53 (Chehab et al. 2000; Hirao et al. 2000, 2002; Shieh et al. 2000; Takai et al. 2002) or in some cases, phosphorylation of PML (Yang et al. 2002). In addition, Chk2-dependent signaling impacts various p53-dependent or p53-independent checkpoints in response to -irradiation (Hirao et al. 2000, 2002; Bartek et al. 2001; Falck et al. 2001; Brown et al. 2002; Jack et al. 2002; McGowan 2002; Takai et al. 2002; Yang et al. 2002). Both Chk2 and p53 mutations have been implicated in the development of Li-Fraumeni syndrome (Malkin et al. 1990; Srivastava et al. 1990; Bell et al. 1999). Unlike p53-deficient mice, Chk2-deficient mice are not susceptible to the spontaneous development of tumors (Hirao et al. 2000, 2002). However, the loss of Chk2 may compromise a "gatekeeper" activity that, when defective, facilitates tumorigenesis through the survival and proliferation of cells with compromised genomic integrity (Kinzler and Vogelstein 1997).
In contrast to a previous study (Foray et al. 2003), our findings demonstrate that Brca1 deficiency results in enhanced Chk2 phosphorylation and that Brca1 is not required for Chk2 activation. Although Chk2 has been shown to interact with, phosphorylate, and colocalize with BRCA1 (Lee et al. 2000), this kinase–substrate relationship of Chk2 and Brca1 in a DNA-damage signaling pathway implies an upstream function of Chk2 with respect to Brca1 and cannot explain the cellular rescue of Brca1 deficiency. Our finding supports a model in which inactivation of Chk2 and Brca1 are cooperative and not epistatic in breast tumorigenesis. This finding seemingly contradicts previous epidemiological studies in which Chk2 and BRCA1 were ascribed to function in the same pathway. A Chk2-truncating variant (Chk21100delC) that has been found to be associated with low-penetrance breast cancer susceptibility appears to be epistatic with respect to BRCA1 mutations (Meijers-Heijboer et al. 2002; Vahteristo et al. 2002). However, separate analysis of Chk2 mutations in human breast cancer found an increased frequency of Chk2 mutations together with p53 mutations in BRCA1-associated breast cancers compared with sporadic breast cancers (Sullivan et al. 2002). Furthermore, the associated loss of Chk2 expression in some breast tumors (Sullivan et al. 2002) suggests a potential role for epigenetic changes that could lead to the loss of Chk2 function and cooperation with BRCA1 mutations in neoplastic transformation.
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Brca1fl5-6 conditional mutant mice (Mak et al. 2000) were intercrossed with LckCre transgenic mice (Jackson Laboratory) or WapCre transgenic mice (Jackson Laboratory) to obtain tBrca1–/– or mBrca1–/– mice, respectively. Subsequent crosses were performed using Chk2–/– mice (Hirao et al. 2002), Eµ-bcl-2-36 transgenic mice (Strasser et al. 1991), and p53–/– mice (Taconic). All mice studied were in a mixed 129/J x C57BL/6 genetic background and were genotyped by PCR (primer sequences and PCR conditions available upon request). Experimental cohorts were derived from littermates obtained from double-heterozygote breeders. Statistical significance of survival curves was assessed using the log-rank test. All experiments were performed in compliance with the Ontario Cancer Institute animal care committee guidelines.
Flow cytometry
Thymocytes, spleen, and lymph nodes were harvested from mice 5–8 wk of age and stained with the following monoclonal antibodies (Pharmingen): anti-CD4, anti-CD8, anti-Thy 1, anti-TCR
, and anti-B220. Fluorescence-activated cell sorting (FACS) analyses were performed using a FACS Calibur and cells were sorted using a FACSVantage flow cytometer (Becton Dickinson).
Apoptosis and activation assays
Thymocytes were either untreated or -irradiated and cultured for 8 h or 24 h prior to harvesting. Apoptosis was monitored by FACS analysis using Annexin V-FITC and propidium iodide (R&D systems). Purified T cells obtained by cell sorting were activated with anti-CD3 (10 µg/mL immobilized) plus anti-CD28 (1 µg/mL), or anti-CD3 (10 µg/mL immobilized) plus murine IL-2 (100 units/mL, Biosource International) and proliferation assessed as described (Mak et al. 2000). Cell-cycle analysis was performed on anti-CD3 + IL2 activated Thy 1+ splenocytes using a BrdU flow kit (Pharmingen).
Western analysis
Western analysis was performed on thymocyte protein lysates using antibodies reactive to murine p53 (CM5, Novocastra), p21 (C-19, Santa Cruz), bax (N-20, Santa Cruz), actin (Sigma), and an affinity-purified rabbit polyclonal antibody raised against amino acids 81–95 of murine Chk2.
Cytogenetic analysis
Peripheral T cells were grown on plate-bound anti-CD3 for 24 h, followed by a 2-d culture in the presence of murine IL-2 (50 units/mL, Biosource International). The activated cells were treated with colcemid (Sigma) for 2 h and prepared for karyotyping (Dracopoli 1999).
Histology
Tumors and major organs were fixed in formalin and paraffin sections were stained with haematoxylin and eosin (H&E) according to standard procedures. Tumor sections were stained with anti-Keratin (Chemicon).
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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Article published online ahead of print. Article and publication date are at http://www.genesdev.org/cgi/doi/10.1101/gad.1192704.
5 Present address: Department of Pharmacology, University of Toronto, Toronto, Ontario M5S 1A8, Canada. 
6 These authors contributed equally to this work.
E-MAIL rhakem{at}uhnres.utoronto.ca; FAX (416) 204-2277.
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