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Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California,1 The Center for Blood Research, The Children's Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts2
Received 19 May 2004/ Returned for modification 29 June 2004/ Accepted 12 October 2004
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ABSTRACT |
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INTRODUCTION |
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-irradiation and multisystemic defects, including growth retardation, abolished germ cell development, immunodeficiency, and greatly increased cancer risk (43). As a protein kinase, ATM functions by phosphorylating and activating a number of DNA repair and checkpoint proteins, including p53, NBS1, H2AX, 53BP1, Brca1, Smc1, and Chk2 (43).
Immediately after DNA DSB damage, histone H2AX is phosphorylated at the C-terminal Ser residues (Ser136 and Ser139) (40). This phosphorylation, called -H2AX, can be detected within minutes after the introduction of DSBs and is involved in the recruitment of other known components of DNA repair, including Brca1, NBS1/Mre11/Rad50, and 53BP1, to the sites of the DNA damage (11, 41, 48). -H2AX is mainly mediated by ATM after DNA DSB damage, suggesting that H2AX is a downstream mediator of ATM function (6, 20). H2AX is also required for the recruitment of 53BP1 to sites of DNA DSB damage, and 53BP1 appears to be an upstream activator of ATM (11, 39, 48). Therefore, it remains unclear whether H2AX plays any role in activating ATM after DNA DSB damage.
NBS1, the gene product mutated in Nijmegen breakage syndrome (NBS) patients, is the p95 component of the Mre11 complex that forms foci at the sites of DNA DSBs (8, 36, 47). The importance of NBS1 in DNA repair is indicated by the multisystemic defects observed in NBS patients, including growth retardation, immunodeficiency, and increased lymphoid malignancies (46). In addition, NBS cells are hypersensitive to ionizing radiation and defective in intra-S and G2/M checkpoints. Targeted disruption of the N terminus of NBS1 in mice recapitulates most of the systemic and cellular defects observed in NBS patients (25, 50). The functional link between ATM and NBS1 is suggested by the large panel of defects shared by NBS1 and ATM mutations and is further demonstrated by the findings that ATM can phosphorylate and activate NBS1 at a consensus site, Ser343 (21, 30, 51, 55). Therefore, NBS1 appears to function as a downstream mediator of ATM function. Recent studies show that mutation of the Mre11 complex or NBS1 leads to impaired ATM activation in human cell lines, indicating a role for NBS1 in activating ATM after DNA DSB damage (9, 23, 39, 45). Since the disruption of this NBS1 function could account for the A-T-related defects observed in NBS patients, the contribution of the impaired adapter function of NBS1 in mediating ATM activities to the A-T-related defects in NBS1 mutant mice and human patients remains to be established.
The functional interaction between NBS1 and H2AX is complex. H2AX is required for the irradiation-induced focus formation of NBS1 after DNA DSB damage (11). In addition, direct interaction between -H2AX and NBS1 suggests a mechanism to recruit NBS1 to the sites of DNA DSB damage (27). These findings suggest that H2AX functions upstream of NBS1 in DNA damage response. However, while the defects observed in either H2AX or NBS1 mutant mice are shared by ATM mutant mice, H2AX and NBS1 mutant mice exhibit certain distinct phenotypes (4, 12, 25, 50, 52). For example, while spermatogenesis is abolished in H2AX-deficient mice, oogenesis failure is only observed in NBS1 mutant mice. Therefore, NBS1 and H2AX might have distinct roles in DNA damage responses.
It has been well established that ATM is required for the activation of p53 responses to DNA DSB damage in human and mouse cells (26, 49, 53, 54). ATM can phosphorylate p53 at Ser15 (corresponding to Ser18 of mouse p53), and this phosphorylation activates p53 responses to DNA DSB damage (13, 14, 17, 28). While it has been well established that ATM is required to activate p53-dependent cell cycle G1 arrest upon DNA DSB damage, it remains unclear whether H2AX is involved in mediating this ATM function after DNA DSB damage. We employed NBS1, H2AX, and p53 mutant mice to investigate the functional interactions among ATM, NBS1, H2AX, and p53 in DNA damage responses, maintenance of genetic stability, and tumor suppression.
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MATERIALS AND METHODS |
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Ionizing radiation (IR) treatment of MEFs and cell cycle analysis.
MEFs were synchronized at the cell cycle G0 phase by serum starvation in medium containing 0.1% fetal bovine serum for 96 h as described previously (54). The G0-synchronized MEFs were trypsinized and irradiated in suspension with a 137Cs -ray source. The irradiated and mock-treated MEFs were plated in 10-cm-diameter plates at a density of 1 million cells/plate in normal MEF growth medium supplemented with 10 µM bromodeoxyuridine. After 24 h of bromodeoxyuridine labeling, the cells were harvested and fixed in 70% ethanol, and the cell cycle profile was analyzed as described previously (53).
Analysis of p53-dependent apoptosis in mouse thymocytes. Thymuses were recovered from 1- to 2-month-old mice of various genotypes, and single-cell thymocyte suspensions were treated with increasing doses of IR as described previously (15). The percentage of apoptotic cells 10 h after IR was analyzed by staining with annexin V as described previously (15).
Western blot analysis of levels of proteins and protein phosphorylation. Protein extracts from 4 x 105 embryonic stem cells or MEFs or 5 x 106 thymocytes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 6 to 15% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with various primary antibodies and subsequently incubated with horseradish peroxidase-conjugated secondary antibody and developed with Enhanced Chemiluminescence PLUS (Amersham Pharmacia Biotech) or a Pierce SuperSignal kit. To determine the amount of protein in each lane, the filter was stripped and probed with a rabbit polyclonal antibody against actin (Santa Cruz Biotechnology, Inc.). Phosphospecific antibody against ATM was purchased from Rockland. Phosphospecific antibody against p53 was purchased from Cell Signaling Technology.
Real-time PCR analysis. Total RNA was isolated from MEFs or thymocytes with the combination of Trizol (Invitrogen) and RNAeasy RNA Cleanup (QIAGEN). RNA was treated with RNase-free DNase (Roche Diagnostics) for 20 min at room temperature before being reverse transcribed using Superscript II reverse transcriptase (Invitrogen). Real-time PCR was performed on an ABI 7000 machine with SYBR Green PCR Master Mix (Applied Biotechnology). PCR conditions consisted of a 10-min hot start at 95°C, followed by 40 cycles of 30 s at 95°C and 1 min at 61°C. The average threshold cycle for each gene was determined from triplicate reactions, and the levels of gene expression relative to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were determined as previously described (5). The primers used were described previously (13).
Generation and analysis of NBS1m/m) H2AX–/– and NBS1m/m p53–/– mice. The ATM–/–, p53–/–, NBS1 mutant/mutant (NBS1m/m), and H2AX–/– mice were described previously (4, 24, 25, 52). Because NBS1m/m females are sterile, p53+/– mice were bred with NBS1m/m males to generate p53+/– NBS1+/m mice, which were intercrossed to generate p53–/– NBS1m/m mice. The health status of various mutant mice was monitored once every 2 days. The dead or dying mice were dissected and examined for tumors. The H2AX–/– males were sterile (4), and H2AX–/– NBS1+/m female and H2AX+/– NBS1–/– males were intercrossed to screen for homozygous mutant embryos.
Analysis of chromosomes. Pretumor thymocytes were cultured in RPM-1 medium supplemented with 12 ng of phorbol myristate acetate/ml and 0.25 µg of ionomycin/ml, and metaphases were prepared after 2 to 3 days of culture when the thymocytes reached the log phase of proliferation. Tumor cells were prepared for spectral karyotyping (SKY) analysis by culturing them in RPM-1 medium supplemented with 10% fetal calf serum, antibiotics, 100 µM ß-mercaptoethanol, and 100 U of interleukin-2/ml for 2 to 3 days until they reached log-phase growth. Thymocytes and tumor cells were treating with 0.05 µg of Colcemid/ml for 2.5 h prior to standard metaphase dropping. Metaphases from pretumor thymocytes were stained with DAPI (4',6'-diamidino-2-phenylindole) and examined. SKY analysis of chromosomes was performed as described previously (19). Chromosomes were analyzed using a Nikon Eclipse microscope equipped with an Applied Spectral Imaging charge-coupled device camera and 40 and 63x objectives.
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RESULTS |
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-H2AX is normal in NBS1m/m cells after IR (Fig. 3D, E, and F). Since NBS1m/m cells expressed an N-terminally truncated NBS1 protein at low levels (25), these findings indicated that the N-terminal domain of NBS1 is not required for the activation of ATM and ATM-dependent phosphorylation of p53 and H2AX after DNA DSB damage.
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Synthetic lethality of NBS1 and H2AX mutations. Since NBS1, but not H2AX, is required for ATM-dependent phosphorylation of Chk2 (20), these findings suggested some distinct functions of H2AX and NBS1 in mediating ATM-dependent activities. In addition, NBS1m/m and H2AX–/– mice developed some distinct defects (4, 12, 25, 50). In support of the notion that NBS1 and H2AX have distinct roles in DNA damage responses, while both NBS1m/m and H2AX–/– mice are viable, NBS1m/m H2AX–/– mice were embryonic lethal (Table 1). The embryonic lethality occurred during early embryonic development, since no homozygous mutant embryos could be detected at embryonic day 12 (Table 1). H2AX haploid deficiency has been shown to increase cancer predisposition in the p53–/– background (4, 10). Therefore, we monitored and compared tumorigenesis in 34 sets of NBS1m/m and NBS1m/m H2AX+/– mice that were >6 months of age. No increased tumorigenesis was observed in NBS1m/m H2AX+/– mice.
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, a predictor of global chromosomal translocations in mouse thymocytes (25, 31). The thymocytes were analyzed by flow cytometry for their patterns of CD4 and CD8 expression, as well as their cell sizes, to ensure that they were not tumor cells. While little interchromosomal rearrangement can be detected in wild-type thymocytes, interchromosomal rearrangements involving these TCR loci were observed in all ATM–/– and NBS1m/m thymocytes analyzed (Fig. 4A and B). However, similar to wild-type thymocytes, little interchromosomal rearrangement was detected in thymocytes derived from 10 H2AX–/– mice and 10 p53–/– mice (Fig. 4A and B and data not shown).
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NBS1 and p53 have synergistic roles in tumor suppression. ATM–/–, NBS1m/m, and p53–/– mice all developed thymic lymphomas (3, 16, 18, 24, 25, 52). However, ATM–/– mice developed thymic lymphomas much faster than NBS1m/m and p53–/– mice and uniformly died from thymic lymphomas by 6 months of age (3, 52). These findings suggest that ATM-dependent activation of p53 and NBS1 might play synergistic roles in tumor suppression. Consistent with this notion, p53-dependent p21 induction and cell cycle G1 arrest were both normal in NBS1m/m MEFs after various doses of IR (Fig. 5A and data not shown). In addition, p53-dependent apoptosis was modestly reduced in NBS1m/m thymocytes after IR compared with that in wild-type thymocytes (Fig. 5B). To further test the functional interactions of NBS1 and p53 in tumor suppression, NBS1m/m p53–/– mice were generated and monitored for tumorigenesis. Similar to ATM–/– mice, NBS1m/m p53–/– mice developed thymic lymphomas much faster than NBS1m/m and p53–/– mice, and all NBS1m/m p53–/– mice died from thymic lymphomas by 5 months of age (Fig. 5C). Therefore, NBS1 and p53 function synergistically in the suppression of thymic lymphomas.
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DISCUSSION |
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Impaired ATM activation and p53 phosphorylation have been reported in human NBS cell lines (9, 23, 39, 45). However, we found normal phosphorylation of ATM and p53 in our NBS1 mutant mice. The apparent discrepancy between our findings in NBS1 mutant mice and the human cell lines is likely due to the nature of NBS1 mutation and the expression levels of the truncated NBS1 in these two systems. Cells from NBS patients express an N-terminal 26-kDa NBS1 protein fragment and an N-terminally truncated 70-kDa NBS1 protein at low levels (34). In NBS1m/m mice, the N terminus (codons 1 to 194) of NBS1, which contains the FHA and BRCT domains, is disrupted (25). Therefore, while the 26-kDa N-terminal NBS1 protein fragment should not be produced in our NBS1 mutant mice, the N-terminally truncated NBS1 protein of 
70 kDa is expressed at reduced levels in the cells derived from the mutant mice (25). The discrepancy in ATM activation between NBS1 mutant mice and NBS patients could be due to the different expression levels of the 70-kDa N-terminally truncated NBS1 protein. Alternatively, since the 26-kDa N-terminal NBS1 fragment is expressed only in some human NBS cells, it is also possible that the 26-kDa N-terminal NBS1 fragment plays a dominant-negative role in suppressing ATM activation after DNA DSB damage.
The finding of normal ATM activation in NBS1m/m mice after DNA DSB damage extends the observation by Horejsi et al. that the N-terminal FHA domain of NBS1 is not required for ATM activation and indicates that neither the FHA nor the BRCT domain of NBS1 is required for ATM activation (23). In addition, consistent with the finding that NBS1 plays important roles in mediating ATM activities (22, 45), this finding indicates that the A-T-related defects observed in NBS1 mutant mice are mainly due to the disruption of the adapter function of NBS1 in mediating ATM activities, such as the phosphorylation of Chk2.
Our studies also provide an improved understanding of the functional interactions among H2AX, NBS1, and p53. An earlier report showed that H2AX is required for the irradiation-induced focus formation of NBS1 after DNA DSB damage, suggesting that H2AX and NBS1 function in overlapping pathways in the cellular responses to DNA DSB damage (11). However, several lines of evidence presented here and elsewhere indicate that NBS1 and H2AX also function in distinct pathways in DNA damage responses. In this context, NBS1, but not H2AX, is required for ATM-dependent Chk2 phosphorylation after DNA DSB damage (Fig. 3) (7, 20). In addition, NBS1, but not H2AX, is required for the ATM-dependent suppression of interchromosomal rearrangement, indicating that NBS1 and H2AX have distinct roles in DNA damage responses. In support of this notion, the chromosomal abnormalities observed in the thymic tumors derived from NBS1m/m and NBS1m/m p53–/– mice are different from those derived from H2AX–/– p53–/– mice (4). The distinct roles of H2AX and NBS1 in DNA repair could also account for the synthetic lethality of NBS1 mutation and H2AX mutation.
ATM is required to activate p53 responses to DNA DSB damage (42). Our finding that H2AX and NBS1 are not important for p53 responses to DNA DSB damage indicates that H2AX and NBS1 function largely in distinct pathways of p53 in DNA DSB damage responses. In the context of H2AX–/– cells, increased basal levels of p53 activities, possibly due to the presence of endogenous genetic instability, might account for the very modest cancer risk in these mutant mice. Consistent with these notions, inactivation of p53 greatly facilitates tumorigenesis in H2AX–/– mice and our NBS1 mutant mice, as well as independently generated NBS1 mutant mice (4, 10, 50). Considering the greatly increased genetic instability in H2AX and NBS1 mutant mice, these findings indicate that p53 can efficiently suppress tumorigenesis in the presence of significant endogenous chromosomal abnormalities. Therefore, cancer predisposition in ATM–/– mice, in which both p53 inactivation and genetic instability are present, is greatly increased compared with that in NBS1 mutant mice.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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