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DNA Damage and Cellular Stress Responses

p53-Independent Apoptosis Disrupts Early Organogenesis in Embryos Lacking Both Ataxia-Telangiectasia Mutated and Prkdc

¹ Program in Developmental Biology, Hospital for Sick Children Research Institute and Departments of ² Immunology and ³ Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada

Requests for reprints: Cynthia J. Guidos, Program in Developmental Biology, Hospital for Sick Children Research Institute, Toronto Medical Discovery Tower Building (East Tower), Room 14-312, 101 College Street, Toronto, Ontario, Canada M5G 1L7. Phone: 416-813-5026; Fax: 416-813-8823. E-mail: cynthia.guidos{at}sickkids.ca

	Abstract

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
The ataxia-telangiectasia mutated (ATM) protein and the nonhomologous end-joining (NHEJ) pathway play crucial roles in sensing and repairing DNA double-strand breaks in postnatal cells. However, each pathway is dispensable for early embryogenesis. Loss of both ATM and Prkdc/Ku is synthetically lethal, but neither the developmental processes perturbed nor the mechanisms of lethality have been determined by previous reports. Here, we show that ATM and Prkdc collaborate to maintain genomic stability during gastrulation and early organogenesis, a period of rapid proliferation and hypersensitivity to DNA damage. At E7.5 to E8.5, ATM^–/–Prkdc^scid/scid embryos displayed normal proliferation indices but exhibited excessive apoptosis and elevated expression of Ser¹⁵-phosphorylated p53. Thus, this crucial regulatory residue of p53 can be phosphorylated in the absence of ATM or Prkdc. However, loss of p53 did not abrogate or delay embryonic lethality, revealing that apoptosis is p53 independent in these in ATM^–/–Prkdc^scid/scid embryos. Because mice with combined disruptions of ATM and other NHEJ components (ligase IV, Artemis) are viable, our data suggest a novel NHEJ-independent function for Prkdc/Ku that is required to complete early embryogenesis in the absence of ATM. (Mol Cancer Res 2006;4(5):311–8)

	Introduction

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
In mammals, multiple DNA double-strand breaks (DSB) repair and DNA damage response pathways collaborate to limit the propagation of damaged chromosomes, thus maintaining genomic stability (1-4). Members of the phosphoinositide 3-kinase–related kinase (PI3K) family play key roles in initiating cellular responses to various kinds of DNA damage (1, 5, 6). Chief among these are the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and Rad3-related (ATR) proteins, which phosphorylate overlapping sets of checkpoint effector proteins to delay cell cycle progression or induce apoptosis following DNA damage. ATR responds to processed DSB that arise at stalled replication forks or UV-induced DNA lesions to induce arrest cells at the G₂-M phase transition. In contrast, ATM rapidly induces G₁-S, intra-S phase, and G₂-M cell cycle checkpoints when it senses even small numbers of unprocessed DSB (5, 6). A key effector of ATM and ATR-mediated DNA damage responses is the p53 tumor suppressor protein, which can arrest the cell cycle or induce apoptosis (7). Repair of DSB can be accomplished by either homologous recombination or nonhomologous end-joining (NHEJ). In mammalian cells, homologous recombination predominates in the S-G₂-M phases of the cell cycle, whereas NHEJ predominates in G₁ (8). NHEJ proteins are ubiquitously expressed and include the DNA-dependent protein kinase (Prkdc), also a PI3K family member, as well as Ku70 and Ku80, Artemis, XRCC4, and DNA ligase IV (Lig4).

Recent studies suggest that early embryonic and postnatal cells have different requirements for DNA damage pathways regulated by PI3K. Homozygous mutations in ATR or its checkpoint effector kinase, Chk1, cause early embryonic lethality, whereas mice lacking ATM or its major effector kinase, Chk2, are born at normal Mendelian ratios (5, 9). Mice with homozygous disruptions of the Prkdc, Ku70, Ku80, or Artemis NHEJ genes are viable; however, homozygous loss of the XRCC4 or Lig4 NHEJ genes causes late embryonic lethality (E16-E18). This lethality is accompanied by massive apoptosis of postmitotic neurons that can be rescued by p53 or ATM deficiency (10-13). Thus, the XRCC4 and Lig4 NHEJ proteins play unique and essential roles during embryonic neurogenesis, but neither ATM nor NHEJ components are required for DSB sensing or DSB repair during early embryogenesis.

Given that neither ATM nor NHEJ proteins, such as Prkdc and Ku, are required for embryogenesis, the embryonic lethality that results from combined genetic disruption of ATM and Prkdc or Ku was unexpected (10, 14). In the course of our studies to identify potential roles of ATM during V(D)J recombination, we also observed synthetic lethality of ATM and Prkdc loss-of-function mutations. Because p53^–/–Prkdc^scid/scid mice are born at expected frequencies viable (15, 16), these observations suggest that ATM has p53-independent functions during embryogenesis that can be accomplished by Prkdc when ATM is absent. To define these functions, we undertook an analysis of the developmental stage and mechanisms by which ATM^–/–Prkdc^scid/scid embryos die. Our data show that ATM and Prkdc have partially overlapping functions that are essential during gastrulation and early organogenesis, the morphogenetic process that occurs between E6.5-E7.5 (in mice) to produce endoderm, mesoderm, and ectoderm.

	Results

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
ATM^–/–Prkdc^scid/scid Embryos Die Early in Embryogenesis
The combined disruption of ATM and Prkdc was synthetically lethal because no ATM^–/–Prkdc^scid/scid mice were observed in a total of 160 viable progeny from ATM^+/–Prkdc^scid/scid intercrosses. Therefore, we did a series of timed matings and genotyped embryos on different days of gestation to determine the time of intrauterine death (Table 1 ). We recovered near Mendelian frequencies of ATM^–/–Prkdc^scid/scid embryos at E7.5 to E9.5 (Table 1), but they were abnormally small and showed delayed development relative to littermate ATM^+/–Prkdc^scid/scid and ATM^+/+Prkdc^scid/scid embryos (data not shown). By E9.5, the gross morphology of ATM^–/–Prkdc^scid/scid embryos was highly abnormal (Fig. 1A ). Subsequently, no ATM^–/–Prkdc^scid/scid embryos were observed at E12.5 to E14.5. However, resorbed double-mutant embryos were observed at near-expected Mendelian frequencies during this timeframe. These data show that ATM^–/–Prkdc^scid/scid embryos can develop to midgestation but they succumb to postimplantation death soon afterwards.

View larger version (64K): [in this window] [in a new window]   FIGURE 1. Morphologic and histologic assessment of ATM–/–Prkdcscid/scid embryos. A. Embryos were dissected, fixed for whole mount imaging, and genotyped by PCR. i, normal morphology of an ATM+/–Prkdcscid/scid embryo is representative of all nine ATM+/+Prkdcscid/scid or ATM+/–Prkdcscid/scid embryos from this litter. At E9.5, normal developmental hallmarks include closure of the neural tube with extensive forebrain (br) development, the presence of forelimb buds (fl), and the formation of 16 to 20 pairs of somites (s). ii to iv, representative example of an ATM–/–Prkdcscid/scid embryo (similar to two others in this litter) showing stunted growth and highly abnormal morphology. Only rudimentary head folds (hf) were seen. Note the different magnification of (i) versus (ii-iv). B and C. Sagittal sections of H&E-stained embryos from timed matings (ATM+/–Prkdcscid/scid intercrosses) showing normal and abnormal morphology from the same litter at E7.5 (B) and E8.5 (C). B. i, normal size and morphology in the extraembryonic tissues (exe) and the embryo proper (e) that has undergone gastrulation producing all germ layers, endoderm (en), mesoderm (m), and ectoderm (ec). Primitive head folds. ii, in contrast, an embryo with abnormal morphology is severely delayed in both size and maturation. C. i, well-developed neural folds (nf), a primitive heart (h), and at least four somites (s) in an embryo with normal morphology. ii, an embryo with abnormal morphology is severely delayed in size and organogenesis has not occurred.

Normal Proliferation but Excessive Apoptosis in ATM^–/–Prkdc^scid/scid Embryos
The absence of Prkdc disrupts NHEJ, leading to the accumulation of DSB, which can activate p53-dependent checkpoints (15). To determine if ATM deficiency prevented activation of DNA damage checkpoint(s), ATM^–/–Prkdc^scid/scid embryos were examined for evidence of activation of cell cycle arrest or apoptosis. Proliferation was assessed in embryos produced from ATM^+/–Prkdc^scid/scid intercrosses using a 5-bromo-2'-deoxyuridine (BrdUrd) incorporation assay. Pregnant females were injected with BrdUrd, which is incorporated during DNA synthesis, and sacrificed 90 minutes later. Immunohistochemistry with an anti-BrdUrd antibody was then done on sectioned embryos at E7.5 and E8.5 to determine the frequency of cells in S phase. Proliferation indices were calculated as the percentage of BrdUrd-positive nuclei relative to the total number of nuclei. In agreement with previous studies (17, 18), we found that E7.5 embryos had high proliferative indices. Importantly, embryos with normal versus abnormal morphology had similar proliferation indices at E7.5 and E8.5 (Fig. 2A ). At E7.5, proliferation indices in extraembryonic regions were similar between normal (55 ± 7%) and abnormal (43 ± 21%) embryos (Fig. 2A). However, at E8.5, proliferation indices in this region of abnormal embryos were highly variable and generally reduced (36 ± 23%) compared with normal embryos (71 ± 8%; Fig. 2A). We suggest that this difference may be a secondary effect due to compromised viability in embryonic tissues rather than a proliferation defect per se (see below). Collectively, these findings show that during the early postgastrulation period, abnormal embryos from ATM^+/–Prkdc^scid/scid intercrosses proliferated to the same extent as normal embryos. Thus, excessive activation of a cell cycle checkpoint is not the underlying mechanism responsible for the embryonic lethality.

View larger version (59K): [in this window] [in a new window]   FIGURE 2. Frequencies of proliferating and apoptotic cells in morphologically normal and abnormal embryos from timed matings of ATM+/–Prkdcscid/scid intercrosses. A. Left, BrdUrd immunostaining of sagittal sections from E7.5 embryos with normal (i) and abnormal (ii) morphology. Sections were counterstained with hematoxylin. The boundary between the extraembryonic tissue and embryonic region. Right, proliferation indices of morphologically normal ( and ) and abnormal ( and ) embryos and extraembryonic tissues at E7.5 and E8.5. B. 4',6-Diamidino-2-phenylindole (blue; i and ii) versus TUNEL (green; iii and iv) staining of embryos with normal (i and iii) and abnormal (ii and iv) morphology at E7.5. Scatter graphs, percentage of TUNEL+ nuclei in morphologically normal ( and ) and abnormal ( and ) embryos at E7.5 and E8.5. C. An adjacent H&E stained section from the mutant embryo seen in (B) (ii) shows several pycnotic nuclei (arrows and enlarged area) consistent with apoptosis (i, ii).

Increased Levels of p53 and Phosphoserine-15 p53 in Abnormal Embryos from ATM^+/–Prkdc^scid/scid Intercrosses
Phosphorylation of p53 on Ser¹⁵ by ATM, ATR, or Prkdc enhances p53-mediated transcriptional activation of checkpoint effectors, such as p21 and Bax (5). To determine whether ATM- and Prkdc-independent activation of p53 might explain the excessive apoptosis we observed in ATM^–/–Prkdc^scid/scid mice, we examined levels of total and phosphoserine-15 p53 in embryonic sections. At E7.5 and E8.5, morphologically normal embryos expressed uniformly low levels of p53 in both extraembryonic and embryonic regions (Fig. 3 ). In contrast, morphologically abnormal embryos exhibited intense p53-specific cellular staining in a patchy distribution (Fig. 3). To determine if abnormal embryos expressed high levels of activated p53, immunohistochemistry was done using a p53 phosphoserine-15-specific antibody. At E7.5, embryos with abnormal morphology exhibited phosphoserine-15 p53 staining, whereas no staining was seen in embryos with normal morphology (Fig. 3). Because immunohistochemistry with antibodies to total p53 and phosphoserine-15 p53 was done on adjacent sections of the same embryo, these data suggested that only a fraction of the p53⁺ cells in abnormal embryos contained significant levels of activated p53. The low amounts of phosphoserine-15 p53 may reflect variation in the timing and duration of Ser¹⁵ phosphorylation in response to DNA damage. Collectively, these findings show that phosphorylation of the Ser¹⁵ residue of p53 can occur in the absence of both ATM and Prkdc.

View larger version (101K): [in this window] [in a new window]   FIGURE 3. Levels of total and Ser15 phosphorylated p53 protein, Bax, and p21 in embryos from ATM+/–Prkdcscid/scid intercrosses. Sagittal sections of embryos with normal and abnormal morphology at E7.5 were stained with polyclonal antibodies recognizing total p53, Ser15 phosphorylated p53, p21, and Bax and counterstained with hematoxylin. A greater frequency of p21-positive cells was consistently seen in the extraembryonic (exe) region compared with embyonic (e) regions. Robust p53 and Ser15 phosphorylated p53 levels and p21 can be seen in our positive control, irradiated thymus (500 cGy). Bax expression levels were the same in normal and abnormal E7.5 embryos, compared with negative controls (no primary antibody) in adjacent embryo sections.

p53 Deficiency Does Not Rescue Embryonic Lethality in ATM^–/–Prkdc^scid/scid Mice
Although in situ histochemical studies did not provide evidence of p53-dependent Bax transcription in ATM^–/–Prkdc^scid/scid embryos, p53 has been shown to induce apoptosis in a Bax-independent fashion (22). Therefore, we took a genetic approach to determine if activation of the p53 checkpoint was required to cause premature death of ATM^–/–Prkdc^scid/scid embryos. p53^–/–ATM^+/–Prkdc^scid/scid males were mated with p53^+/–ATM^+/–Prkdc^scid/scid females to generate p53-deficient ATM^–/–Prkdc^scid/scid progeny (expected frequency of p53^–/–ATM^–/–Prkdc^scid/scid, 12.5%). No live p53^–/–ATM^–/–Prkdc^scid/scid pups were identified in >149 live born pups analyzed (Table 2 ). Timed matings at E8.5 were done to determine if loss of p53 function delayed the lethality in ATM^–/–Prkdc^scid/scid embryos (Table 2). Of 47 E8.5 embryos examined, 3 p53^–/–ATM^–/–Prkdc^scid/scid embryos were observed, and all 3 had severe growth and maturational delays consistent with the mutant phenotype seen in E8.5 ATM^–/–Prkdc^scid/scid embryos. Thus, disruption of the p53 DNA damage checkpoint did not rescue or delay embryonic lethality in ATM^–/–Prkdc^scid/scid mice, suggesting that the early postgastrulation death of these embryos results from p53-independent apoptosis.

	Discussion

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

DNA damage responses operate under special cell cycle conditions during gastrulation. First, the cell cycles are extremely rapid and include only brief periods of interphase (17), likely explaining the reliance of early embryos on homologous recombination for repairing DSB. Moreover, embryonic, but not extraembryonic, cells are hypersensitive to DNA-damaging agents between E6.5 and E7.5 but not during periods immediately preceding or following gastrulation (18). Thus, during gastrulation, embryonic cells undergo rapid proliferation and are hypersensitive to DNA damage. Surprisingly, in response to ionizing radiation, gastrulating embryos do not arrest in G₁ but instead undergo apoptosis (18).

At E7.5, the double-mutant embryos had developed all three germ layers but were abnormally small; however, we did not observe G₁ arrest of ATM^–/–Prkdc^scid/scid embryos (Fig. 2A). Rather, combined loss of ATM and Prkdc caused high levels of spontaneous apoptosis in embryonic regions (Fig. 2B). In contrast, the apoptotic indices were low in extraembryonic tissues. These data suggested that gastrulation had occurred, but excessive apoptosis limited embryo growth during this process. Although double-mutant embryos survived for a few more days in utero, they remained abnormally small and did not undergo organogenesis. Collectively, these data suggest that loss of both ATM and Prkdc triggered a DNA damage response that eliminated early embryonic cells by apoptosis without arresting DNA synthesis. Because embryogenesis seemed relatively normal until E7.5, we suggest that excessive apoptosis during gastrulation and the early postgastrulation phase resulted in arrested organogenesis causing embryonic lethality.

We found that ATM^+/–Prkdc^scid/scid E7.5 embryos ubiquitously expressed low levels of p53, but Ser¹⁵ phosphorylation of the protein was absent (Fig. 3). These observations agree with previous studies, showing that p53 is widely expressed although relatively inactive during early embryogenesis (24). However, embryos with abnormal morphology from ATM^+/–Prkdc^scid/scid intercrosses had increased levels of p53 protein expression in both embryonic and extraembryonic tissues, and a fraction was phosphorylated on Ser¹⁵. Although we also observed some p53 Ser¹⁵ phosphorylation in extraembryonic regions, apoptotic indices were low (<0.5%), and these tissues are quite resistant to DNA damage-induced apoptosis (18). Thus, extraembryonic defects are unlikely to be responsible for the developmental failure of the double-mutant embryos.

Phosphorylation of p53 on Ser¹⁵ depends on activation of phosphoinositide 3-kinase–related kinase family members by DNA damage and is thought to correlate with activation of p53-dependent DNA damage responses (5). However, we found that two transcriptional targets of p53, Bax and p21, were not differentially expressed in ATM^–/–Prkdc^scid/scid embryos (Fig. 3). Moreover, we found that p53 deficiency did not rescue or delay embryonic lethality in ATM^–/–Prkdc^scid/scid embryos (Table 2). Thus, the early embryonic lethality is not due to excessive activation of the p53 DNA damage checkpoint in ATM^–/–Prkdc^scid/scid mice, in contrast to XRCC4 or Lig4-deficient mice. Rather, loss of ATM and Prkdc caused p53-independent apoptosis at E7.5 to E9.5.

The reasons that ATM^–/–Prkdc^scid/scid embryos undergo excessive apoptosis remain to be determined. Although they have not activated a G₁ checkpoint, our data do not exclude the possibility that they have activated intra-S phase or G₂-M checkpoints possibly in an ATR-dependent fashion (2). However, the intra-S phase checkpoint only transiently delays S-phase progression, and if damage is unrepaired, the cells arrest later at the G₂-M checkpoint. Thus, prolonged activation of a G₂-M checkpoint could result in p53-independent death. Alternatively, loss of ATM may abrogate the G₂-M checkpoint, allowing cells to enter into mitosis with unrepaired DNA damage. This situation generally causes arrest at the metaphase-anaphase transition, causing p53-independent death by mitotic catastrophe (3).

Finally, it is possible that the excessive apoptosis of ATM^–/–Prkdc^scid/scid embryos is due to loss of a prosurvival pathway. ATM is thought to maintain neuronal homeostasis by protecting postmitotic neurons from apoptosis (25, 26). ATM is also required for hematopoietic stem cell maintenance by preventing accumulation of reactive oxygen species (27). In support of this notion, we have shown recently that ATM regulates a Bcl-2-independent survival pathway that protects postmitotic thymocytes from apoptosis as they undergo T-cell receptor rearrangement.⁴ We speculate that an ATM-dependent prosurvival pathway could be particularly crucial when NHEJ-mediated DSB repair is disrupted by loss of Prkdc or Ku proteins. Furthermore, a recent study showed that ATM and Prkdc can act in a p53-independent manner to oppose the apoptotic response to DNA damage by activating the nuclear factor-B survival pathway (28). Moreover, ATM transcriptionally regulates a cluster of prosurvival genes, including nuclear factor-B and its regulators (28-30). Further analyses of ATM/Prkdc double-mutant embryos may provide novel insights into ATM-dependent prosurvival pathways that may be operative during early embryogenesis.

	Materials and Methods

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
ATM^–/– Severe Combined Immunodeficient Mice
ATM-deficient severe combined immunodeficient (SCID) mice were bred and housed in specific pathogen-free conditions at the Hospital for Sick Children animal facility (Toronto, Ontario, Canada). 129SvEv.ATM^+/– mice (31) were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred to C.B-17.Prkdc^scid/scid mice to produce ATM^+/–Prkdc^scid/+ mice that were subsequently backcrossed to C.B-17.Prkdc^scid/scid mice to generate ATM^+/–Prkdc^scid/scid mice. ATM^+/–Prkdc^scid/scid mice were intercrossed to generate the embryos examined in this study. Tail DNA was PCR amplified to genotype ATM and Prkdc alleles.

Because no live births of ATM^–/–Prkdc^scid/scid mice were observed, timed matings from E7.5 to E14.5 were done to determine the stage of intrauterine death. Genomic DNA from yolk sacs or embryos was isolated for genotyping by PCR amplification using standard conditions.

p53-Deficient ATM^–/– SCID Mice
ATM^+/–p53^+/+Prkdc^scid/scid females were crossed to ATM^+/+p53^–/–Prkdc^scid/scid males (15). The resulting ATM^+/–p53^+/–Prkdc^scid/scid progeny was then intercrossed. In addition, an alternative breeding strategy mating ATM^+/–p53^+/–Prkdc^scid/scid females and ATM^+/–p53^–/–Prkdc^scid/scid males was used. As expected, ATM^+/– and ATM^+/+p53^–/–Prkdc^scid/scid mice developed pro-B-cell leukemia at 6 to 12 weeks of age (15). Because no live births of ATM^–/–p53^–/–Prkdc^scid/scid mice were observed, timed matings and genotyping of embryos by PCR were done to determine the time of intrauterine death in the context of all three homozygous mutations.

Analysis of Embryonic Lethal Phenotype in ATM^–/–Prkdc^scid/scid Mice
Detection of BrdUrd-Labeled Cells. To examine ATM^–/–Prkdc^scid/scid embryos proliferation indices, BrdUrd labeling and immunohistochemistry was done. BrdUrd (Sigma, St. Louis, MO) in PBS (5 mg/mL) was injected (100 µg/g of body weight) i.p. into pregnant females (E7.5 and E8.5), which were sacrificed 90 minutes after injection. The decidua were dissected, fixed in 4% paraformaldehyde or 70% ethanol, and processed for immunohistochemistry. Paraffin-embedded serial sagittal sections were dewaxed and rehydrated according to standard procedures. Slides were then incubated with a 1:20 dilution of an anti-BrdUrd antibody (BD PharMingen, San Jose, CA) and the Vectastain avidin-biotin complex method kit (Vector Laboratories, Burlingame, CA). BrdUrd-positive nuclei were detected using 3,3'-diaminobenzidine, the slides were counterstained with hematoxylin, and BrdUrd-positive cells were counted. Results were expressed as a ratio of BrdUrd-positive nuclei to the total number of nuclei. Images were obtained using a Zeiss (Toronto, Ontario, Canada) microscope and Adobe Photoshop 7.0 software.

TUNEL Assay. TUNEL assays were done to examine if mutant embryos had excessive levels of apoptosis. The fluorescent ApoAlert DNA Fragmentation Assay kit (Clontech, Palo Alto, CA) was used to detect apoptotic cells. Modifications to the manufacturer's instructions included the use of high temperature antigen retrieval with a 0.01 mol/L sodium citrate (pH 6.0) buffer and 4',6-diamidino-2-phenylindole mounting media (Vector Laboratories) to counterstain the nuclei. The total number of TUNEL-positive cells in both extraembryonic and embryonic tissue was quantitated and divided by the total number of 4',6-diamidino-2-phenylindole–stained nuclei to generate a percentage of apoptosis score.

Immunohistochemistry on Sectioned Embryos. Paraffin-embedded slides of embryos within decidua were prepared as described above. Sections were dewaxed in xylene and rehydrated in a decreasing ethanol series. H&E staining was done according to standard procedures. All sections were treated for antigen retrieval as described above. Immunohistochemistry was done on slides blocked in PBS with 5% goat serum for 30 minutes. Primary antibodies used included anti-p53 [CM5 (1:500), Novocastra Laboratories, Newcastle upon Tyne, United Kingdom], p53 phosphospecific Ser¹⁵ [Ab-3 (1:100), Oncogene Research, San Diego, CA], Bax (1:250; PharMingen, San Diego, CA), and p21 [M-19 (1:200), Santa Cruz Biotechnology, Santa Cruz, CA] and detected with a secondary biotinylated anti-rabbit IgG antibody (1:200; Vector Laboratories). Immunoreactivity was detected with Vectastain avidin-biotin complex method kit and 3,3'-diaminobenzidine and counterstained with hematoxylin. Thymic lobes were removed from 6- to 8-week-old C57BL6 mice 2 hours after receiving 500 cGy from a ¹³⁷Cs irradiator and paraffin embedded, treated for antigen retrieval, and used to validate DNA damage-induced staining for p53, p53 phosphospecific Ser¹⁵, p21, and Bax.

	Acknowledgements

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
We thank Drs. Janet Rossant and C.C. Hui (Program in Developmental Biology, Hospital for Sick Children) for assistance in histology techniques.

	Notes

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Grant support: Canadian Institutes of Health Research and the National Cancer Institute (NCI) of Canada (R.A. Gladdy); Scientist Awards from the Canadian Institutes of Health Research and the NCI of Canada (C.J. Guidos and J.S. Danska); Principal Investigator of the Canadian Genetic Disease Network (J.S. Danska); and NCI of Canada (with funds from the Canadian Cancer Society).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Current address for R.A. Gladdy: Department of Surgery, University of Toronto, 100 College Street, Toronto, Ontario, Canada M5G 1L5; T. Kunath: Institute for Stem Cell Research, University of Edinburgh, Roger Land Building, King's Buildings, West Mains Road, Edinburgh EH9 3JQ, United Kingdom.

⁴ I.R. Matei et al., submitted for publication.

Received 12/10/05; revised 2/12/06; accepted 2/22/06.

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