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The molecular pathways leading from genomic instability to cellular senescence and/or cell death remain incompletely characterized. Using mouse embryonic fibroblasts with constitutively increased DNA damage due to the absence of the full-length form of the tumor suppressor Brca1 (Brca1Δ11/ Δ11), we show that deletion of p53 binding protein 1 (53BP1) selectivity abrogates senescence and cell death stimulated by reduced Brca1 activity. Furthermore, the embryonic lethality induced by Brca1 mutation can be alleviated by 53BP1 deletion. Adult Brca1Δ11/ Δ1153BP1-/- manifest constitutively high levels of genomic instability, yet age relatively normally with a surprisingly low incidence of overall tumor formation. Together, these in vitro and in vivo data suggests that 53BP1 is specifically required for the development of premature senescence and apoptosis induced by Brca1 deficiency. These observations may have important implications for Brca1 mediated tumor formation as well as for the molecular pathway leading from genomic instability to organismal aging.
DNA damage induces a range of cellular responses ranging from growth arrest, to the induction of senescence or apoptosis. It is generally believed that in the setting of such damage, the lack of cellular proliferation or the loss of cell viability provides an efficient tumor suppressor mechanism. While inhibiting cancer, the accumulation of senescent and apoptotic cells can also contribute and potentially augment the rate of organismal aging. Indeed, a number of mammalian models of chronic DNA damage and genomic instability are characterized by exhibiting both a tumor-prone and accelerated aging phenotype (Chen et al., 2007; Lombard et al., 2005; Serrano and Blasco, 2007).
Brca1 is an important checkpoint and DNA damage repair gene that is required for maintaining genomic intergrity. We have previously described a mouse model of constitutive DNA damage in which exon 11 of the Brca1 gene has been deleted resulting in the absence of the full length Brca1 isoform. Similar to Brca1 null mice, the Brca1Δ11/ Δ11 mice in general die in utero, although this embryonic lethality happens slightly later in development in the Brca1Δ11/ Δ11 animals than in the null embryos (Hakem et al., 1997; Ludwig et al., 1997; Xu et al., 2001). Further analysis of Brca1Δ11/ Δ11 embryos has demonstrated that there is a significant increase in both spontaneous cell death and cellular senescence (Cao et al., 2006; Cao et al., 2003; Xu et al., 2001). Although the observed embryonic lethality of Brca1Δ11/ Δ11 mice can be rescued by deletion of one allele of p53, the resulting Brca1Δ11/ Δ11 p53+/- mice exhibit a high rate of spontaneous tumor formation in multiple organs (Xu et al., 2001). Detailed molecular analysis of these malignancies has demonstrated that essentially all of the tumors that arise are accompanied by a loss of heterozygosity in the p53 locus (Brodie et al., 2001; Cao et al., 2006). In addition to this tumor prone phenotype, the Brca1Δ11/ Δ11 p53+/- mice also exhibit many features that are consistent with accelerated aging (Cao et al., 2006; Cao et al., 2003).
The embryonic lethality of the Brca1Δ11/ Δ11 mice can also be rescued by deletion of certain components of the DNA damage pathway (DDR) including ATM and Chk2 (Cao et al., 2006). Here, we have further characterized the in vitro and in vivo pathways activated in Brca1Δ11/ Δ11 mice. Our results identify 53BP1 as essential for mediating cell death and senescence induced by Brca1 deficiency but dispensable for apoptosis and senescence induced by a variety of other DNA damaging agents.
Mouse embryonic fibroblasts (MEFs) isolated from Brca1Δ11/ Δ11 mice are known to undergo rapid premature senescence in culture since these cells manifest a constitutive increase in DNA damage secondary to reduced Brca1 activity (Cao et al., 2006; Cao et al., 2003). Using these MEFs along with a candidate gene approach, we sought to identify genes specifically required for Brca1 mediated premature senescence. In culture, Brca1Δ11/ Δ11 MEFs rapidly manifest a senescent morphology (Figure 1A) and exhibit positive staining for senescence associated β-galactosidase (SA-βgal). By passage 5, nearly 75% of MEFs isolated from Brca1Δ11/ Δ11 mice were SA-βgal positive (Figure 1B).
Based on our previous observations (Cao et al., 2006; Cao et al., 2003; Xu et al., 2001), we asked whether perturbation of various DDR or cell cycle regulatory components could rescue Brca1Δ11/ Δ11 mediated premature senescence. MEFs were prepared from crosses between Brca1+/ Δ11 mice and various other animals containing targeted deletions of genes within these identified pathways. As noted in Figure 1B, although Chk2 deletion can rescue the Brca1Δ11/ Δ11 embryonic lethality (Cao et al., 2006), Brca1Δ11/ Δ11Chk2-/- MEFs appear to prematurely senesce at the same elevated rate as Brca1Δ11/ Δ11 cells. In contrast, deletion of one allele of p53 resulted in a small reduction in the rate of Brca1Δ11/ Δ11 induced senescence, while deletion of both p53 alleles appeared to completely abrogate the observed Brca1Δ11/ Δ11 stimulated premature senescence. Such results were not unexpected as p53 deletion abrogates senescence induced by numerous stimuli (Riley et al., 2008; Rodier et al., 2007). Similar analysis with MEFs containing targeted deletions of ATM, Chk1, H2AX, p21, PTEN, Gadd45a, p19ARF, and Parp1 revealed that none of these gene deletions could rescue premature senescence caused by reduced Brca1 activity (unpublished observations). In contrast, we observed that Brca1Δ11/ Δ11 MEFs lacking 53BP1, a DNA damage response and p53 binding protein (Adams and Carpenter, 2006), were seemingly resistant to the observed accelerated senescence (Figure 1B). Similarly, while Brca1Δ11/ Δ11 MEFs appeared to have increased levels of p53 and evidence for increased p53 activity (Figure 1C), these biochemical changes were not evident in Brca1Δ11/ Δ1153BP1-/- MEFs.
To assess whether 53BP1 could also rescue Brca1Δ11/ Δ11 mediated senescence in vivo, we took advantage of previous observations that developing Brca1Δ11/ Δ11 embryos manifest an intense senescence mediated growth arrest (Cao et al., 2006; Cao et al., 2003). Consistent with those previous reports, Brca1Δ11/ Δ11 embryos were smaller than wild type embryos and stained intensely positive for SA-βgal (Figure 1D). This premature embryonic senescence was noticeably absent in Brca1Δ11/ Δ1153BP1-/- embryos. Interestingly, Brca1Δ11/ Δ11 embryos exhibited evidence for constitutive 53BP1 activation (Figure 1E). This response appeared relatively specific as a similar activation was not evident in H2AX-/- embryos, even though H2AX is also required for the maintenance of genomic stability (Celeste et al., 2002). Previous results have documented that 53BP1 recruitment to DNA requires specific alterations in histone lysine methylation (Botuyan et al., 2006; Huyen et al., 2004). Consistent with these observations, we observed increased staining for histone H4 dimethylated Lys 20 in Brca1Δ11/ Δ11 embryos but not in H2AX-/- embryos (Figure 1E). Other histone modifications known to act as lower affinity binding sites for 53BP1 were also selectively observed in Brca1Δ11/ Δ11 embryos (Figure S1).
Given that 53BP1 deletion rescued senescence induced by reduced Brca1 activity, we next asked what role 53BP1 played in other pathways leading to cellular senescence. The growth of MEFs in 20% oxygen results in passage dependent accumulation of senescent cells that in rodent fibroblasts appears to be a stress-mediated, ‘culture shock’ like phenomena (Parrinello et al., 2003). As expected, WT MEFs had a passage-dependent increase in senescence and a corresponding increase in p53 levels and activity under these growth conditions (Figure S2). An identical biological and biochemical response was seen in 53BP1-/- MEFs. In contrast, p53-/- MEFs had a complete abrogation of this senescent response (Figure S2). A similar analysis employing other triggers of senescence including oxidative stress with exogenous hydrogen peroxide or gamma-irradiation demonstrated that WT and 53BP1-/- MEFs responded similarly (Figure 1F). In contrast, in p53-/- MEFs, the induction of senescence following these stresses was severely compromised or absent.
The above observations suggest that 53BP1 plays a specific role in Brca1Δ11/ Δ11 mediated senescence. To begin to try and understand the basis of the specificity, we sought to analyze and compare the cellular response to reduced Brca1 activity, to the response observed with other forms of DNA damage. In MEFs, both irradiation and Brca1 deficiency resulted in activation of 53BP1 and H2AX (Figure 1G). As previously described, in both irradiated cells and in cells treated with hydrogen peroxide (Figure S3), these DNA damage foci also appeared to recruit additional factors including RAD51 and MDC1(van Attikum and Gasser, 2009). Interestingly, the recruitment of both RAD51 and MDC1 was not evident in Brca1Δ11/ Δ11 MEFs (Figure 1H). This suggests that the DNA damage foci formed in the setting of reduced Brca1 activity are qualitatively different than what is observed with other DNA damaging stresses.
In addition to inducing cellular senescence, the Brca1Δ11/ Δ11 mutation can also trigger programmed cell death and this is particularly evident within the developing embryo (Cao et al., 2006; Cao et al., 2003; Xu et al., 2001). We therefore analyzed rates of apoptosis in WT, Brca1Δ11/ Δ11 or Brca1Δ11/ Δ11 53BP1-/- embryos. Similar to our observations regarding Brca1Δ11/ Δ11 mediated senescence, deletion of 53BP1 appeared to dramatically rescue Brca1Δ11/ Δ11 mediated cell death (Figure 2A and B).
The ability of 53BP1 deletion to rescue in vivo embryonic senescence and apoptosis stimulated by the lack of Brca1 activity suggested that 53BP1 deletion might rescue the overall embryonic lethality caused by decreased Brca1 activity (Cao et al., 2006; Cao et al., 2003; Xu et al., 2001). Indeed, numerous healthy offspring were obtained in the setting of Brca1Δ11/ Δ11 along with deletion of 53BP1, while we observed no viable offspring of Brca1Δ11/ Δ11 mice containing both copies of 53BP1 and only very rare survival of Brca1Δ11/ Δ11 53BP1+/- mice (Table 1). Again, 53BP1 appeared unique in this role, as we observed no viable Brca1Δ11/ Δ11H2AX-/- mice (Table S1).
The survival of Brca1Δ11/ Δ11 53BP1-/- mice allowed us to assess whether cells from these animals manifested a generalized alteration in their apoptotic threshold. As expected, gamma irradiation triggered a significant increase in apoptosis of WT thymocytes (Figure 2C). This response was unaltered in 53BP1-/- thymocytes, both in terms of the degree of cell death, as well as the DDR signaling pathway triggered by irradiation (Figure 2D). Similarly, thymocytes obtained from Brca1Δ11/ Δ11 53BP1-/- mice had similar biological and biochemical responses to irradiation when compared to WT cells (Figure 2C and D). In contrast, numerous previous studies have demonstrated that thymocytes obtained from either p53-/- or Chk2-/- mice are significantly impaired in their ability to undergo apoptosis following irradiation (Clarke et al., 1993; Hirao et al., 2000; Lowe et al., 1993). A similar analysis in MEFs demonstrated that 53BP1-/- cells had intact and in some cases even an augmented apoptotic response following exposure to hydrogen peroxide, doxorubicin or gamma-irradiation (Figure 2E).
One potential explanation for the ability of 53BP1 deletion to rescue mice expressing Brca1Δ11/ Δ11 is that 53BP1 somehow altered or reduced the level of DNA damage and genomic instability in Brca1Δ11/ Δ11 expressing cells and tissues. To exclude this possibility we analyzed the activation of the DDR pathway in Brca1Δ11/ Δ11 or Brca1Δ11/ Δ1153BP1-/- cells, making use of our previous observation that nuclear foci of γ-H2AX were evident in Brca1Δ11/ Δ11 MEFs (Figure 1G). Overall, the degree of H2AX nuclear foci appeared similar when comparing MEFs (Figure 3A and B) or embryonic tissues (Figure 3C-E) derived from either Brca1Δ11/ Δ11 or Brca1Δ11/ Δ1153BP1-/- mice. Similarly, metaphase spreads derived from Brca1Δ11/ Δ1153BP1-/- adult B cells exhibited a high degree of chromatid and chromosomal breaks (Figure 3F&G).
We next sought to analyze the overall biological consequences of 53BP1 deletion in the setting of the Brca1Δ11/ Δ11 expression. After weaning, Brca1Δ11/ Δ11 53BP1-/- as well as 53BP1-/- mice appeared outwardly healthy (Figure 4A), although by three months of age, mice deficient in 53BP1 exhibited a very modest but significant weight reduction compared to WT mice (Figure S4). Similarly, at a year of age, Brca1Δ11/ Δ1153BP1-/- mice continued to weigh and to appear essentially indistinguishable from WT mice (Figure S5). In contrast, Brca1Δ11/ Δ11 animals rescued by haploinsufficiency of p53, weighed only 70% as much as WT mice at one month of age and approximately 50% of WT animals at 7 months of age (Cao et al., 2006; Cao et al., 2003).
In contrast to Brca1Δ11/ Δ1153BP1-/- mice, by seven months of age, Brca1Δ11/ Δ11p53+/- mice exhibited clear evidence of accelerated aging (Figure 4A). This included among other signs, the development of marked kyphosis, as well as changes in the animal’s coat and overall physical activity. We also observed increased senescence in Brca1Δ11/ Δ11p53+/- tissues when compared to WT or Brca1Δ11/ Δ1153BP1-/- tissues (Figure 4B). Similarly, assessment of the rates of spontaneous apoptosis in rapidly dividing organs such as the intestine revealed that Brca1Δ11/ Δ11 expression triggered significantly increased cell death in p53+/- rescued mice, but not in Brca1Δ11/ Δ1153BP1-/- mice (Figure 4C). Consistent with this increase in cell death, Brca1Δ11/ Δ11p53+/- cells had a constitutive activation of p53 not observed in cells obtained from either 53BP1-/- or Brca1Δ11/ Δ1153BP1-/- mice (Figure S6).
Given that accumulation of senescent cells and increased cell death are both thought to mediate the accelerated aging phenotypes seen in models of chronic DNA damage, we next sought to assess the rate of aging in Brca1Δ11/ Δ11 mice that were rescued by either 53BP1 deletion or by haploinsufficiency of p53. As noted above, the latter mice exhibited increased tissue senescence and apoptosis triggered by Brca1Δ11/ Δ11 expression, while in 53BP1-/- rescued mice, these responses were largely absent. Analysis of skin thickness (Figure 4D) and bone density (Figure S7) revealed that the changes associated with accelerated aging present in Brca1Δ11/ Δ11p53+/- mice, were not evident in Brca1Δ11/ Δ1153BP1-/- animals. This point is reinforced by the analysis of overall lifespan. As opposed to Brca1Δ11/ Δ11p53+/- mice, whose maximal lifespan is roughly one year, nearly 80% of Brca1Δ11/ Δ1153BP1-/- were still alive at 20 months (Figure 4E).
Finally, we have previously reported that Brca1Δ11/ Δ11 mice rescued by homozygous deletion of p53 rapidly develop multiple tumors (Brodie et al., 2001; Cao et al., 2006; Xu et al., 2001). We observed a similar high rate of cancer deaths in Brca1Δ11/ Δ11p53+/- mice, with a median tumor free survival time of approximately 9 months (Figure 4F). In contrast, and somewhat unexpectedly, Brca1Δ11/ Δ11 mice lacking 53BP1 exhibited only a very modest rate of cancer formation (<10%) in the first 20 months of life. Analysis of the tumors that developed in these animals demonstrated an absence of breast cancer; rather the tumor spectrum closely mirrored what has been previously observed in 53BP1 deficient mice with all tumors in the first 18 months consisting of thymic lymphomas (Ward et al., 2003)
In summary, we have demonstrated that 53BP1 is required for the induction of senescence or apoptosis triggered by reduced Brca1 activity. Interestingly, although 53BP1 deletion abrogates these responses in the setting of reduced Brca1 activity, the absence of 53BP1 does not appear to modulate the induction of senescence or apoptosis triggered by other stimuli. This selectivity stands in contrast to other genetic manipulations such as deletion of p53 that block senescence and apoptosis mediated not only by Brca1 but for a wide range of DNA damaging stresses. Although the precise mechanism underlying this selectivity is unknown, the overall composition of DNA damage foci formed in Brca1 deficient cells are significantly different from those foci formed in the setting of exogenous DNA damage. Both irradiation and oxidative stress recruit a number of proteins such as Rad51 and MDC1 that are not seen in the foci formed in Brca1Δ11/ Δ11 cells. We speculate that in the setting of these exogenous and perhaps stronger DNA damaging stresses, the recruitment of additional factors somehow lessens the ultimate requirement for 53BP1 expression.
There is a growing realization that the accumulation of senescent and apoptotic cells might contribute to organismal aging, as well as serving as a barrier to tumor formation (Bartkova et al., 2005; Bartkova et al., 2006; Braig et al., 2005; Chen et al., 2005; Collado et al., 2005; Di Micco et al., 2006; Gorgoulis et al., 2005; Michaloglou et al., 2005). One of the interesting observations from the current study is the extended lifespan and near wild type appearance of Brca1Δ11/ Δ11 53BP1-/- mice. The relative absence of various progeriod phenotypes (e.g. kyphosis, reduced skin thickness, osteoporosis, etc.), as well as the extended overall lifespan, suggests that blocking cellular senescence and cell death mitigates the accelerated aging phenotype seen in this model of DNA damage. Perhaps more surprising are the observations that Brca1Δ11/ Δ1153BP1-/- mice exhibit constitutive high levels of genomic instability, yet still do not manifest a marked increase in tumor formation over the first 20 months of life. In previous examples where the Brca1Δ11/ Δ11 mice have been rescued from embryonic lethality, there was an extraordinary high rate of subsequent malignancies. These high rates of tumor incidence include a near 100% incidence in Brca1Δ11/ Δ11 p53-/- mice by 3 months of age (Bachelier et al., 2003; Brodie et al., 2001; Xu et al., 2001) and a greater than 70% incidence of breast cancer in female Brca1Δ11/ Δ11Chk2-/- mice by 16 months of age (Cao et al., 2006). One potential explanation for the relatively low levels of tumor formation in the Brca1Δ11/ Δ11 53BP1-/- mice compared to previous results is that Brca1Δ11/ Δ11 53BP1-/- cells appear to maintain an intact senescent and apoptotic response to various other stresses. In contrast, following exposure to a wide range of DNA damaging stresses, mice deficient in either Chk2 or p53 are known to have a generalized and widespread impairment in their apoptotic and\or senescent response.
Recent evidence has suggested that there may some degree of commonality in the mechanisms underlying cancer and aging (DePinho, 2000; Finkel et al., 2007; Serrano and Blasco, 2007). As we have discussed, the accumulation of senescent cells may be beneficial in blocking tumorgenesis, yet harmful by contributing to organismal aging. As such, our current results provides a potentially instructive example of a model of DNA damage and genomic instability where it would appear that one can selectively ameliorate age-dependent pathologies without significantly increasing cancer rates. Further refinement of our understanding of the molecular pathways leading from DNA damage to cell death and senescence may ultimately allow for new strategies that directly combat age-related phenotypes without substantially altering the predisposition for cancer.
53BP1 (Ward et al., 2003), H2AX (Celeste et al., 2002), Chk2 (Takai et al., 2002), Atm (Barlow et al., 1996) and p53 (Donehower et al., 1992) mice were analyzed alone or where indicated crossed with Brca1+/ Δ11 mice (Xu et al., 2001) to generate double-mutant mice. MEF cells were derived from E14.5 embryos using standard methods and subsequently cultured in DMEM supplemented with 15% FBS.
For routine histology analysis, tissues were fixed in 10% formalin, blocked in paraffin, sectioned, stained with hematoxylin and eosin and subsequently examined by light microscopy. Antibodies for 53BP1 (Novus, 1:000), γ-H2AX (Upstate, 1:000), Rad51 (Santa Cruz, 1:200), MDC1 (Abcam, 1:200), H4K20me3, H4K20Me2 and H3K79 (all Abcam at 1:500) were employed for immunohistological analysis. Detection of the primary antibody was performed using the Histomouse TM Kit (ZYMED) according to the manufacturer’s instruction.
Western blot analysis was performed according to standard procedures using ECL detection (Amersham). The following primary antibodies were used: p53 (BD Pharmingen), p21 (Santa Cruz Biotechnology), ATM p-Ser1987 (BD Pharmingen), Chk2 (Upstate), p53 p-Ser15 (Cell Signalling) and SMC1 (Novus Biologicals).
For SA-βgal analysis of MEFs, cells were washed with PBS (pH 7.2), fixed with 0.5% glutaraldehyde in PBS (pH 7.2) and processed as previously described (Dimri et al., 1995). For in vivo SA-βgal analysis, frozen sections were obtained and subsequently fixed in 1% formalin/PBS for 1 min prior to staining, while embryo senescence was determined as previously described (Cao et al., 2003). Where indicated, early passage MEF cells were treated with 20 μM hydrogen peroxide or with 10 Gy γ-IR, cultured for an additional 7 days and then assessed for SA-βgal staining.
For assessment of cell death in vivo, we performed TUNEL assays on tissue sections on embryos at day E18 using the TUNEL kit (Chemicon). Measurement of thymocyte cell death under basal conditions and following irradiation were performed using propidium iodide and a fluorogenic caspase-3 substrate as previously described (Komoriya et al., 2000). Apoptosis of MEF cells treated with hydrogen peroxide (50 μM), doxorubicin (200 ng/ml) or 15 Gy γ-IR was assessed 24 hrs after treatment using propidium iodide (Sigma) staining and subsequent analysis for sub-G0/G1 events on a Becton-Dickinson FACSCalibur.
Bone density was assessed by an X-ray dose of 15 kV for 100 seconds using a Faxitron X-ray apparatus. The analysis of genomic instability was performed on metaphases prepared from splenic B cells, enriched by CD43 MACS depletion (Miltenyi). Isolated B cells were cultured for 60hrs with LPS and IL-4. The metaphases were prepared by standard protocols (hypotonic lysis with fixation in 3:1 methanol:acetic acid) on colcemid-arrested cells as previously described (Callen et al., 2007).
We are grateful to N. Motoyama for the generous gift of Chk2-/- mice. This work was supported by NIH Intramural funds and a grant from the Ellison Medical Foundation (TF).
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