BRCA2 is required for neurogenesis and suppression of medulloblastoma
2007; Springer Nature; Volume: 26; Issue: 11 Linguagem: Inglês
10.1038/sj.emboj.7601703
ISSN1460-2075
AutoresPierre‐Olivier Frappart, Youngsoo Lee, Jayne M. Lamont, Peter J. McKinnon,
Tópico(s)Telomeres, Telomerase, and Senescence
ResumoArticle3 May 2007free access BRCA2 is required for neurogenesis and suppression of medulloblastoma Pierre-Olivier Frappart Pierre-Olivier Frappart Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Youngsoo Lee Youngsoo Lee Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Jayne Lamont Jayne Lamont Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Peter J McKinnon Corresponding Author Peter J McKinnon Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Pierre-Olivier Frappart Pierre-Olivier Frappart Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Youngsoo Lee Youngsoo Lee Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Jayne Lamont Jayne Lamont Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Peter J McKinnon Corresponding Author Peter J McKinnon Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Author Information Pierre-Olivier Frappart1, Youngsoo Lee1, Jayne Lamont1 and Peter J McKinnon 1 1Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA *Corresponding author. Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA. Tel.: +1 901 495 2700; Fax: +1 901 526 2907; E-mail: [email protected] The EMBO Journal (2007)26:2732-2742https://doi.org/10.1038/sj.emboj.7601703 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Defective DNA damage responses in the nervous system can result in neurodegeneration or tumorigenesis. Despite the importance of DNA damage signalling, the neural function of many critical DNA repair factors is unclear. BRCA2 is necessary for homologous recombination repair of DNA and the prevention of diseases including Fanconi Anemia and cancer. We determined the role of BRCA2 during brain development by inactivating murine Brca2 throughout neural tissues. In striking contrast to early embryonic lethality after germ-line inactivation, Brca2LoxP/LoxP;Nestin-cre mice were viable. However, Brca2 loss profoundly affected neurogenesis, particularly during embryonic and postnatal neural development. These neurological defects arose from DNA damage as Brca2LoxP/LoxP;Nestin-cre mice showed extensive γH2AX in neural tissue and p53 deficiency restored brain histology but lead to rapid formation of medulloblastoma brain tumors. In contrast, loss of the Atm kinase did not markedly attenuate apoptosis after Brca2 loss, but did partially restore cerebellar morphology, supporting a genomic surveillance function for ATM during neurogenesis. These data illustrate the importance of Brca2 during nervous system development and underscore the tissue-specific requirements for DNA repair factors. Introduction Within the nervous system, appropriate responses to DNA damage are required to maintain homeostasis and prevent disease (McMurray, 2005; Lee and McKinnon, 2006). DNA double-strand breaks (DSBs) trigger a signalling cascade that leads to repair and resolution of the break, or as is frequent in the developing nervous system, apoptosis. The repair of DNA DSBs occurs via two mechanistically distinct pathways non-homologous end-joining (NHEJ) or homologous recombination (HR). Each pathway involves a distinct repertoire of repair enzymes and associated proteins. HR requires a group of RAD51-related proteins and a variety of other factors, including BRCA2, to ensure high-fidelity DNA repair using an undamaged homologous DNA template to replace an adjacent damaged one (Thompson and Schild, 2002; West, 2003). In contrast, NHEJ facilitates direct modification and ligation of the two DNA ends at the DSB. Efficient NHEJ requires among other factors, KU heterodimers (KU 70 and KU 80), DNA-PKcs, DNA ligase IV (LIG4) and XRCC4 (Lees-Miller and Meek, 2003; Lieber et al, 2003; Mills et al, 2003; O'Driscoll and Jeggo, 2006). Inactivation of either of these pathways in mice can lead to embryonic lethality or tumor development. Broad insights illuminating DNA repair and nervous system development have been gained from mice in which gene targeting has disabled various DNA repair pathways (Friedberg and Meira, 2006). For example, inactivation of NHEJ in the mouse can lead to defective neurogenesis or brain tumors depending on the particular gene disruption and genetic background (Gao et al, 1998, 2000; Lee and McKinnon, 2002; Yan et al, 2006). Disruption of HR can also affect neural development (Deans et al, 2000; Orii et al, 2006), although when some key HR components such as Brca2 or Rad51 are inactivated, the result is early embryonic lethality during gastrulation (before neural development) (Lim and Hasty, 1996; Tsuzuki et al, 1996; Ludwig et al, 1997; Sharan et al, 1997; Suzuki et al, 1997). Moreover, DNA repair activity exhibits a clear tissue- and cell type specificity, as in the nervous system, HR only functions in proliferating cells, while NHEJ is particularly important in differentiated neural cells (Orii et al, 2006). Together, these data show that DNA DSB repair pathways operate in a complementary manner during neural development. BRCA2 has a key role in HR and substantial DNA repair-associated defects result from its inactivation, including DNA damage hypersensitivity, chromosomal rearrangements and defective mammalian gametogenesis (Sharan et al, 1997; Tutt and Ashworth, 2002; Yang et al, 2002; Daniels et al, 2004). BRCA2 is a large protein of 3418 amino acids that physically interacts through its carboxyl terminus with RAD51, a protein essential for HR, and is responsible for the translocation of RAD51 to sites of DNA damage processing (Powell et al, 2002; Pellegrini and Venkitaraman, 2004; Shivji and Venkitaraman, 2004; Yang et al, 2005). BRCA2 has also been implicated in cell-cycle regulation via interaction with BRAF35 or Smad3 (Marmorstein et al, 2001; Preobrazhenska et al, 2002), and a role during an intra-S phase checkpoint has also been reported (Taniguchi et al, 2002). Brca2 functions as a tumor suppressor, as its loss confers susceptibility to breast, ovarian and brain tumors (Hughes-Davies et al, 2003; Offit et al, 2003; Shivji and Venkitaraman, 2004). The BRCA2-binding protein BALB2 is important for the tumor suppressor function of BRCA2 (Xia et al, 2006; Erkko et al, 2007; Rahman et al, 2007). Biallelic hypomorphic mutations of BRCA2 are also responsible for some cases of Fanconi Anemia (FA), a rare autosomal recessive cancer susceptibility syndrome characterized by congenital abnormalities, progressive bone narrow failure and cellular hypersensitivity to DNA cross-linking agents (Kennedy and D'Andrea, 2005; Taniguchi and D'Andrea, 2006). FA results from inactivating mutations in any one of a multiprotein complex that functions during DNA repair (D'Andrea, 2003; Kennedy and D'Andrea, 2005). FA patients carrying BRCA2 mutations exhibit a more severe phenotype compared with other FA groups, including predisposition to medulloblastoma brain tumors (Offit et al, 2003; Shivji and Venkitaraman, 2004). Defects in the BRCA2 partner-protein PALB2 can also result in FA (Reid et al, 2007; Xia et al, 2007). Given the importance of BRCA2 during HR and the lethality associated with germ-line inactivation, we used conditional gene inactivation via Cre/LoxP technology to determine the requirement for Brca2 during neural development. Inactivation of Brca2 resulted in microcephaly associated with defects in neurogenesis particularly during cerebellar development. Loss of Brca2 also led to medulloblastoma when p53 was disabled. Thus, Brca2 fulfils a critical role during nervous system development, highlighting the tissue-specific requirements for DNA repair during neurogenesis. Results Inactivation of Brca2 leads to microcephaly and cerebellar defects To determine the requirement for Brca2 during development of the nervous system, we used a Brca2 conditional mutant allele in which exon 11 was flanked by LoxP sites (Jonkers et al, 2001). To inactivate Brca2, we used Cre that was driven by the Nestin promoter, resulting in expression throughout the central and peripheral nervous systems from embryonic day 10.5 (Dahlstrand et al, 1995; Frappart et al, 2005). Similar to other reports using this Nestin-cre transgenic line (Graus-Porta et al, 2001; Frappart et al, 2005), we observed efficient deletion of Brca2 throughout the nervous system, as determined using genomic DNA or RNA extracted from the mutant cerebrum or cerebellum (Supplementary Figure 1). In contrast to early embryonic lethality after germ-line inactivation of Brca2 (Ludwig et al, 1997; Sharan et al, 1997; Suzuki et al, 1997), Brca2LoxP/LoxP;Nestin-cre mice (hereafter referred to as Brca2Nes-cre) were viable and were relatively normal in overall appearance, size and behavior. However, inactivation of Brca2 led to microcephaly as Brca2Nes-cre brains were smaller and weighed significantly less (P<0.0001) compared with littermate controls (Brca2LoxP/LoxP or Brca2+/+;Nestin-cre littermates; hereafter referred to as Brca2Ctrl) (Figure 1A–C). All Brca2Nes-cre brain structures were proportionally affected, including the hippocampus, cortex and olfactory bulb (Figure 1A). However, despite the effect on brain size, overall neural development was relatively normal and normal cortical lamination was present (Figure 1A). Notably, the Brca2-deficient cerebellum exhibited stunted foliation and lobule morphology (Figure 1D), and ectopic localization of some Purkinje cells was observed (Figure 1E), suggesting that migration defects might occur during Brca2Nes-cre cerebellar development. Although Cre expression in the brain can lead to microcephaly in some situations (Forni et al, 2006), on no occasion did we observe this with Nestin-cre mice that were wild type (WT) or heterozygous for the Brca2 mutant allele. Figure 1.Brca2 loss leads to neurogenesis defects in a p53-dependent manner. (A) The Brca2LoxPLoxP;Nestin-cre (Brca2Nes-cre) mutant brain is substantially smaller than Brca2+/+;Nestin-cre (Brca2Ctrl) controls, although general morphology is intact. The cortex and the hippocampus (hippo) maintain relatively normal laminar structure (arrows). The cerebellum is markedly smaller in the mutant. (B) Reduced brain size at P21 in Brca2Nes-cre mice compared with that in Brca2Ctrl mice. Rescue of cerebella size occurs in the Brca2Nes-cre;p53−/− mice. (C) The relative brain weight of P7 Brca2Nes-cre and Brca2Nes-cre;p53−/− compared with Brca2+/+;Nes-cre (Brca2Ctrl) shows rescue by p53 loss. (D) H&E staining of Brca2LoxP/LoxP (Brca2Ctrl), Brca2Nes-cre and Brca2Nes-cre;p53−/− brain sections at P21 reveals size reduction of the mutant cerebellum compared with WT (magnification × 2). Calbindin (D-28K) staining shows the Purkinje cell layer is intact in the Brca2Nes-cre cerebellum (magnification × 40), although ectopic localization of Purkinje cells occurs in some lobules (E, magnification × 20). ML, molecular layer; PC, Purkinje cells; IGL, inner granular layer. Download figure Download PowerPoint As the phenotypic severity of many DNA repair mutant mice are rescued by associated p53 inactivation (Gao et al, 2000; Lee and McKinnon, 2002), we generated Brca2Nes-cre mice in which p53 was also inactivated. We found that p53 inactivation significantly rescued the microcephaly of Brca2-deficient mice (P<0.0001; Figure 1B–D) and contributed to restoration of cerebellar structure (Figure 1D and E);p53 heterozygosity also significantly improved cerebellar development, but had no significant effect on the occurrence of microcephaly (data not shown). Loss of Brca2 activates DNA damage-induced apoptosis As p53 functions to signal DNA damage, we questioned if the phenotypic rescue resulting from p53 loss resulted from a block in DNA damage signalling to activate apoptosis. Therefore, to determine if Brca2 loss in the nervous system resulted in DNA damage, we assessed phosphorylated H2AX (γH2AX) levels in Brca2Nes-cre brains; γH2AX occurs in response to DNA DSBs and is a canonical marker for this type of damage (Rogakou et al, 1998; Fernandez-Capetillo et al, 2004). We found that the cells of the cerebellar external germinal layer (EGL) of mutant mice exhibited increased γH2AX foci (Figure 2A). These foci were concentrated in the outer EGL of the cerebellum that corresponds to the proliferative granule cell progenitors, suggesting that DNA DSBs resulting from Brca2 loss likely occurred during replication. This is consistent with a role for BRCA2 in stabilizing DNA structures at stalled replication forks (Lomonosov et al, 2003). Figure 2.Brca2 loss leads to DNA damage and increased apoptosis in EGL granule cell progenitors. (A) Loss of Brca2 leads to H2AX phosphorylation (γH2AX) in Brca2Nes-cre and Brca2Nes-cre;p53−/− but not control; Brca2Ctrl (Brca2Flox/Flox) P7 cerebellum (magnification × 20); insets show higher magnification of γH2AX (magnification × 40). (B) Compared with control Brca2Ctrl (Brca2+/+;Nestin-cre) P7 cerebellum, apoptosis is widespread throughout the Brca2Nes-cre cerebellum, but is attenuated after associated loss of p53, as assessed by TUNEL or immunostaining for apoptosis-related single-stranded DNA (ssDNA) (magnification × 40). Quantification of TUNEL staining (C) or pyknotic nuclei (D) in Brca2+/+;Nes-cre (Brca2Ctrl) P7 EGL compared with Brca2Nes-cre showed a significant increase in apoptosis (P<0.0001), while apoptosis was attenuated in Brca2Nes-cre;p53−/− tissue. (E) Apoptotic cells with typical nuclear morphology of pyknosis commonly show colocalization with biochemical apoptotic markers (ssDNA immunostaining) (magnification × 100). Download figure Download PowerPoint DNA damage during development of the nervous system can result in apoptosis (Lee and McKinnon, 2006). To determine if the DNA DSBs identified by γ-H2AX resulted in apoptosis, we used TUNEL or apoptosis-associated single-stranded DNA (ssDNA) assays (Frankfurt et al, 1996; Kawarada et al, 1998) to assess the WT and mutant developing cerebellum. We found that Brca2 inactivation resulted in apoptosis in the cerebellar EGL, while co-inactivation of p53 led to a significant decrease of apoptosis (Figure 2B and C). Apoptosis was restricted to proliferative granule cell progenitors and some early differentiating post-mitotic cells (that may have incurred sublethal damage during proliferation) in the Brca2Nes-cre EGL, as demarcated by immunostaining for Tag-1, a marker for premigratory granule cells (Supplementary Figure 2). However, some apoptosis still occurred in Brca2Nes-cre;p53−/− cerebella, indicating that while p53-dependent signalling accounts for most apoptosis in the Brca2-deficient cerebellum, there is some that is p53 independent. To confirm that the apoptosis assays specifically reflect apoptotic cells, we also quantified cells with pyknotic nuclei that are indicative of apoptosis. As shown in Figure 2D, the abundance of pyknotic cells reflects the number found using the TUNEL assay. Under the conditions used here, these distinct assay methods are accurate indicators of apoptosis and do not simply label damaged DNA. Both methods failed to show any positive apoptotic signal in various brain tissues up to 2 h after ionizing radiation treatment despite the presence of substantial levels of γ-H2AX in these tissues (data not shown). Finally, both ssDNA (Figure 2E) and TUNEL (not shown) positive signal were associated with pyknotic cells. Therefore, loss of Brca2 invokes a DNA damage response that can lead to apoptosis during development. Brca2 inactivation promotes increased apoptosis but not proliferation defects While apoptosis results from loss of Brca2, it is possible that proliferation defects also contribute to microcephaly. Therefore, we used bromodeoxyuridine (BrdU) incorporation to quantify proliferation, but found no statistically significant differences in BrdU incorporation between the mutant or WT genotypes at 90 min (Figure 3A) or 6 h (data not shown). Thus, Brca2 loss results in apoptosis, but does not appear to affect granule neuron progenitor proliferation. Figure 3.Apoptosis and proliferation analysis of Brca2Nes-cre cerebella. (A) Analysis of proliferation was determined after BrdU incorporation in control tissue and Brca2Nes-cre;p53+/+ and Brca2Nes-cre;p53−/− EGL. Proliferation is not perturbed by Brca2 loss, as BrdU incorporation is similar between Brca2Nes-cre and Brca2Ctrl. (B) Increased phosphorylated histone H3-positive cells were found in the Brca2Nes-cre and Brca2Nes-cre;p53−/− EGL (P<0.0001). Asterisks indicate significant differences and n indicates the number of different cerebella analyzed from each genotype. Download figure Download PowerPoint Because Brca2-deficient cells in vitro can accumulate in G2 (Patel et al, 1998; Marmorstein et al, 2001), we examined cell-cycle progression using phosphohistone H3-specific immunostaining to identify cells in G2/M. We found a significant increase (P=0.0229) of cells that were immunopositive for phosphorylated histone H3 in Brca2Nes-cre cerebellar EGL compared with control tissue (Figure 3B). We also found that the increase of G2/M cells after Brca2 loss was present in the Brca2Nes-cre;p53−/− EGL, indicating that this cell-cycle block is independent of p53 (Figure 3B). These data are consistent with DNA damaged cells activating a G2 arrest before either DNA repair or apoptosis (Kastan and Bartek, 2004; Sancar et al, 2004). Apoptosis induced by Brca2 loss is present throughout neural development To further assess the effects of Brca2 loss, we analyzed neural development at different developmental stages. We quantified BrdU-positive cells in the cerebellar primordia at E14.5 and similar to postnatal cerebellar development, we did not observe any differences in proliferation between control (Brca2+/+;Nestin-cre) and Brca2Nes-cre at this age (Figure 4A), although apoptosis was increased at this stage and remained elevated through postnatal development (Figure 4B). Figure 4.Developmental analysis of Brca2 loss. Proliferation and apoptosis were determined at various developmental times in Brca2Ctrl and Brca2Nes-cre neural tissue. Analysis of apoptosis was performed using TUNEL and proliferation was determined after analysis of BrdU incorporation. (A) No difference in proliferation was found at early developmental times for the cerebellum. (B) Significantly increased apoptosis (P<0.0001) was found throughout neural development in mutant (Brca2Nes-cre) tissue; Brca2Ctrl was Brca2+/+;Nestin-cre. (C) Representative BrdU staining in the developing E14.5 hindbrain ventricular zone (magnification × 40). (D) Representative TUNEL staining in the developing E14.5 hindbrain ventricular zone (magnification × 40). Download figure Download PowerPoint We also analyzed other brain regions including the E14.5 neuroepithelium of the hindbrain, and again found no difference in proliferation, but there was an increase in apoptosis within this region of the central nervous system (CNS) (Figure 4C and D). Therefore, cell loss from apoptosis most likely accounts for the occurrence of microcephaly present in Brca2Nes-cre animals. Atm deficiency partially restores morphology of the Brca2Nes-cre cerebellum Atm is required for DNA DSB-induced apoptosis in select neural populations (Lee et al, 2000; Sekiguchi et al, 2001; Kruman et al, 2004; Orii et al, 2006), although DNA damage from defective HR does not signal Atm (Orii et al, 2006; Adam et al, 2007). However, in those studies, the HR mutation was germ-line inactivation of Xrcc2, and resulted in early embryonic lethality. This early embryonic lethality precluded analysis of Atm signalling during later neural development. Therefore, to further determine Atm function after disruption of HR, we crossed Brca2 mutant mice with Atm+/− mice and obtained Brca2Nes-cre;Atm−/− animals. Notably, we found that Atm deficiency promoted partial recovery of cerebellar development (Figure 5A–C). However, in contrast to p53 deficiency, Atm loss did not rescue the microcephaly resulting from Brca2 inactivation (Figure 5A–C). While Atm deficiency contributed to a reduction of apoptosis in the EGL (Figure 5D), it did not alter proliferation of the granule cell progenitors (Figure 5D). However, when we measured TUNEL-positive cells in the EGL, the statistical significance (P=0.1219) between the Brca2Nes-cre and the Brca2Nes-cre; Atm−/− mice suggested that Atm loss contributes to the rescue of only a small fraction of the apoptotic cells in the Brca2Nes-cre EGL. This partial rescue of the Brca2Nes-cre cerebellum is consistent with a role for Atm after granule precursors exit the cell cycle, rather than a primary function in proliferating cells (Lee et al, 2001). ATM may therefore act as a backup surveillance to ensure cells containing DNA damage don't become incorporated into mature neural tissue. Figure 5.Atm inactivation restores cerebellar growth but not microcephaly in Brca2Nes-cre mice. (A) Partial rescue of cerebellar development in Brca2Nes-cre mice occurs when Atm is inactivated. (B) Comparison of brain weight at P7 between Brca2+/+;Nes-cre (Brca2Ctrl), Brca2Nes-cre and Brca2Nes-cre;Atm−/− mice shows that loss of Atm does not restore microcephaly, as brain weight between Brca2Flox/Flox (Brca2Ctrl) and Brca2Nes-cre; Atm−/− is still significantly different (P<0.0001). (C) H&E staining of Brca2Flox/Flox (Brca2Ctrl), Brca2Nes-cre and Brca2Nes-cre;Atm−/− of P21 cerebellum sections shows that development is partially restored by Atm deficiency (magnification × 2); calbindin (D-28K) staining reveals the molecular layer (ML), the Purkinje cell layer (PC) and the inner granule layer (IGL), (magnification × 40). (D) Although proliferation is similar in the EGL of P7 Brca2Ctrl, Brca2Nes-cre and Brca2Nes-cre;Atm−/− cerebella, TUNEL is reduced in the post-mitotic region (parentheses) but not the proliferative layer (asterisk) (magnification × 40); n indicates the number of cerebella analyzed. Download figure Download PowerPoint Brca2 deficiency leads to defects in neural progenitor cell self-renewal and proliferation The previous analyses indicate apoptosis is increased after loss of Brca2, leading to defective neural development, and both proliferative progenitor cells and early post-mitotic neurons are affected. To further investigate potential targets of Brca2-deficiency, we examined neurosphere cultures. Neurospheres were established from Brca2Ctrl, Brca2Nes-cre and Brca2Nes-cre;p53−/− brains at E14.5 and P0 (Figure 6A and B). PCR analysis showed that there was efficient gene deletion in E14.5 Brca2Nes-cre neurospheres (data not shown). There was a substantial reduction in the number and size of neurospheres derived from the Brca2Nes-cre brains compared with those from Brca2Ctrl or Brca2Nes-cre;p53−/− brains (Figure 6A and B). When E14.5 neurospheres were cultured, the number of spheres was significantly reduced in Brca2-deficient animals after 7 days in culture (P<0.0001) (Figure 6B) compared with Brca2Ctrl and Brca2Nes-cre;p53−/− animals, potentially indicating less CNS stem/progenitor cells in Brca2-deficient animals, or alternatively, decreased proliferation or survival in culture. The Brca2-deficient neurospheres propagated less readily, as indicated by a smaller number of cells per sphere (Figure 6C). Notably, at later stages, we were also unable to isolate P0 Brca2-deficient neurospheres after two independent attempts (data not shown). However, inactivation of p53 restored the number of Brca2Nes-cre neurospheres and number of cells per neurosphere after 7 days in culture of either E14.5 or P0 neural stem cells (Figure 6B–D). To determine why Brca2-deficient neurospheres were compromised, we analyzed apoptosis and proliferation. Similar to Brca2 loss in vivo, we found that there was increased apoptosis in Brca2Nes-cre neurospheres, but we found reduced BrdU incorporation in neural progenitor cells in vitro (Figure 6E). However, p53 inactivation restored normal proliferation and substantially reduced apoptosis in Brca2Nes-cre;p53−/− neurospheres (Figure 6E and F). The discrepancy between proliferation defects in vitro but not in vivo after Brca2 loss may reflect culture stress activating cell-cycle checkpoints. Alternatively, as stem cells are a minor population, our BrdU assays may have missed this compartment in the tissue we examined. Overall, these data suggest that Brca2Nes-cre neural progenitors undergo increased rates of apoptosis and their loss may additionally contribute to the microcephaly observed in the Brca2Nes-cre nervous system. Figure 6.Analysis of Brca2Nes-cre neural progenitor cells. (A) Morphology of E14.5 Brca2Ctrl, Brca2Nes-cre and Brca2Nes-cre;p53−/− neurospheres after 7 days in culture. (B) Numbers of E14.5 neurospheres after an initial seeding of 2.5 × 105 cells/ml derived from control or mutant embryos. (C) Numbers of cells present in E14.5 neurospheres derived from control or mutant embryos. (D) BrdU and TUNEL staining of E14.5 neural progenitor cells 3 h after BrdU treatment. (E) Quantitation of BrdU-positive cells in the Brca2Nes-cre neural progenitor cell population compared with Brca2Ctrl, and the respective number of TUNEL-positive cells (F). Asterisks indicate statistically significant differences; n indicates the number of individual cell lines analyzed. Download figure Download PowerPoint Brca2 is required to suppress medulloblastoma In the nervous system, inactivation of DNA DSB repair can lead to medulloblastoma (Lee and McKinnon, 2002; Holcomb et al, 2006; Yan et al, 2006), and BRCA2 mutations in FANCD1 are also associated with this brain tumor (Offit et al, 2003). Therefore, to determine if Brca2 functions as a tumor suppressor in the brain, we monitored tumor formation in five experimental groups over a period of 32 weeks: Brca2Ctrl, Brca2Nes-cre, Brca2Nes-cre;p53+/−, p53−/− and Brca2Nes-cre;p53−/−. Although Brca2Nes-cre mice exhibit a significantly shorter lifespan compared with control mice (Figure 7A) (P 80% died by 32 weeks of age. Brca2Nes-cre mice with associated p53−/− or p53+/− mutations succumbed to medulloblastoma. Total animal numbers are indicated. (B) Examples of typical medulloblastomas in Brca2/p53-deficient mice, represented by a dashed line. Analysis of Brca2Nes-cre;p53−/− medulloblastomas using H&E, Ki-67 or synaptophysin (magnification × 20). (C) SKY analysis of tumors showing translocations and genomic rearrangements on chromosome 11 in Brca2Nes-cre;p53+/− tumors. (D) PCR analysis of p53 showing the loss of p53 WT allele in Brca2Nes-cre;p53+/−; T represents tumor and N represents tail DNA from the same animal. Download figure Download PowerPoint Table 1. Incidence, onset and chromosome 11 involvement of medulloblastoma in Brca2/p53-deficient mice Genotype Medulloblastoma/ total animals (incidence) Onset (weeks)±s.d. Tumors and chromosome 11 loss Brca2Ctrl 0/26 (0%) — — Brca2Nes-cre 0/31 (0%) — — P53−/− 0/29 (0%) — — Brca2Nes-cre;p53+/− 34/47 (72%) 20.53±5.64 16/17 (94%) Brca2Nes-cre;p53−/− 19/23 (83%) 13.26±2.86 0/8 (0%) However, from 10 weeks of age onwards, most Brca2Nes-cre;p53−/− mice became moribund with medulloblastoma (n=19/23; Table I and Figure 7A). Although p53−/− mice generally succumb to lymphoid tumors, in no case did we observe medulloblastoma in these mice (n=29; Table I and Figure 7A). Notably, medulloblastoma also occurred in p53 heterozygous mice that were Brca2Nes-cre (n=34/47), although with a significantly increased tumor latency compared with Brca2Nes-cre;p53−/− mice (∼13 weeks versus ∼21 weeks; P<0.0001) (Figure 7A and B; Table I). Consistent with this, arrayCGH or spectral karyotyping (SKY) identified genomic rearrangements of chromosome 11 (on which p53 resides) in Brca2Nes-cre;p53+/− tumors (Figure 7C, Table I and Supplementary Figure 2). SKY analysis showed chromosome 11 translocations involved various other chromosomes (Figure 7C), while arrayCGH showed loss of chromosomal material spanning the region of chromosome 11 containing p53 (Supplementary Figure 2). In contrast, no rearrangements or loss of c
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