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DNA Damage and Cellular Stress Responses

Ataxia-Telangiectasia Mutated Is Not Required for p53 Induction and Apoptosis in Irradiated Epithelial Tissues

Fred Hutchinson Cancer Research Center, Seattle, Washington

Requests for reprints: Christopher J. Kemp, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, C1-015, Seattle, WA 90109-1024. Phone: 206-667-4252; Fax: 206-667-5815. E-mail: cjkemp{at}fhcrc.org

	Abstract

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
The ataxia-telangiectasia mutated (Atm) protein kinase is a central regulator of the cellular response to DNA damage. Although Atm can regulate p53, it is not known if this Atm function varies between tissues. Previous studies showed that the induction of p53 and apoptosis by whole-body ionizing radiation varies greatly between tissue and tumor types, so here we asked if Atm also had a tissue-specific role in the ionizing radiation response. Irradiated Atm-null mice showed impaired p53 induction and apoptosis in thymus, spleen, and brain. In contrast, radiation-induced p53, apoptosis, phosphorylation of Chk2, and G₂-M cell cycle arrest were slightly delayed in Atm^–/– epithelial cells of the small intestine but reached wild-type levels by 4 h. Radiation-induced p53 and apoptosis in Atm^–/– hair follicle epithelial cells were not impaired at any of the time points examined. Thus, Atm is essential for radiation-induced apoptosis in lymphoid tissues but is largely dispensable in epithelial cells. This indicates that marked differences in DNA damage signaling pathways exist between tissues, which could explain some of the tissue-specific phenotypes, especially tumor suppression, associated with Atm deficiency. (Mol Cancer Res 2007;5(12):1312–8)

	Introduction

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Mutation in the ataxia-telangiectasia mutated (Atm) gene, located on chromosome 11q22-23, results in a pleiotropic disorder in humans known as ataxia-telangiectasia (1). Among many symptoms, ataxia-telangiectasia patients have a predisposition to lymphoid and other malignancies and a marked sensitivity to ionizing radiation (IR; ref. 2). Cells from ataxia-telangiectasia patients have a defect in DNA double-strand break repair and show impaired cell cycle checkpoints, increased chromosome instability, and hypersensitivity to IR (3, 4). The Atm gene encodes a 370-kDa protein that has serine/threonine protein kinase activity (4). A region at the COOH terminus of Atm is highly related to the catalytic domain of phosphatidylinositol 3-kinase. This region, as well as an additional conserved COOH-terminal region, is shared by DNA-PK_cs, ATR, and FRAP/TOR, which all belong to the phosphatidylinositol 3-kinase–related protein kinase superfamily (5). Atm is activated by DNA double-strand breaks and phosphorylates several effectors, including Chk2 and p53. Atm phosphorylation of Ser¹⁵ of p53 has been proposed as a mechanism by which DNA damage up-regulates p53 in response to IR (6-8). Mdm-2, which is a feedback regulator of p53, is also phosphorylated in an Atm-dependent manner and this is another mechanism of p53 regulation (9). Collectively, these result in stabilization of p53, increased p53 protein levels, and increased transcription of p53-regulated genes (10). Two key functions of the tumor suppressor p53 are mediating the G₁ cell cycle checkpoint through induction of the cyclin-dependent kinase inhibitor p21 and induction of apoptosis through induction of proapoptotic proteins, such as Puma, Noxa, or Bax (11). In addition, activated Chk2 phosphorylates Cdc25A and Cdc25C, leading to the inactivation of cyclin-dependent kinases and cell cycle arrest in S or G₂-M (12). Although there is agreement that Atm participates in the DNA damage response, and thus has a potential role in tumor suppression, results from experiments with Atm-mutant cells have generated conflicting results about p53 regulation (4, 13, 14). For example, some studies have found Atm cells to be defective in the induction of p53 (15-17), whereas other studies found no defect or a delay in p53 induction (18). The generation of Atm knockout mouse models (19-21) has allowed the examination of the role of Atm in the DNA damage response of cells in their native environment. Studies using these mice have also produced conflicting results. Two groups reported impaired radiation-induced p53 in mouse embryonic fibroblasts and reduced apoptosis in thymocytes from Atm-mutant mice (22, 23), whereas two other groups reported no defect in apoptosis of Atm-null thymocytes (24, 25). Herzog et al. (25) found impaired p53-dependent apoptosis in the developing central nervous system of Atm-null mice. These different results could be due to different radiation doses, time points, or assay methods used.

Understanding the role of Atm in the DNA damage response has increased in relevance with recent reports showing evidence of DNA damage signaling in precancerous lesions (26, 27). In particular, increased H2A.X, a marker of DNA damage, and activation of Atm, Chk2, and p53 were seen in human tumors. The interpretation of these findings was that the DNA damage pathway is constitutively active in premalignant tumors, creating selective pressure against p53, and that this signaling pathway is a barrier to cancer progression (26, 27). However, it is well known that the regulation of p53 by DNA damage varies greatly between tissues and tumor types when assayed in vivo. For example, following whole-body IR, p53 is induced in limited cell types, notably lymphoid cells, epithelial cells within the small intestine, colon, and skin, and several other cell types (28). Some, but not all, of these cell types also undergo p53-dependent apoptosis. The inducibility of p53 also varies greatly between developing autochthonous tumors (29). Mice that had developed tumors of diverse histologic subtypes were given a standard radiation dose and tumors examined at defined time points. Thymic lymphoma cells underwent rapid and extensive p53-dependent apoptosis, whereas p53 induction and apoptosis were essentially undetectable in irradiated premalignant adenomas of the liver and lung. Papillomas of the skin, and adenomas of the breast, and intestine showed an intermediate response. Clearly, the DNA damage, Atm, and p53 circuit could only play a role in tissue biology and tumor suppression if the pathway is functional within that particular tissue or tumor type. The aim of this study was to examine the role of Atm in p53 regulation and apoptosis in multiple tissues in vivo using a standard dose of IR and standard assay methods.

	Results

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Spontaneous levels of apoptosis in the thymus, small intestine, and skin were low and did not differ between Atm-null, heterozygous, and wild-type mice. p53 was undetectable by immunostaining in all tissues examined from all genotypes.

DNA Damage Response in Thymus and Spleen
Atm deficiency in the thymus led to a marked and gene dosage-dependent reduction in IR-induced apoptosis. After 4 Gy IR, the thymic cortex from wild-type mice, containing mostly radiation-sensitive CD4⁺ CD8⁺ cells, showed increasing levels of apoptosis over the 10 h examined (Fig. 1A ). In Atm-null mice, however, the number of apoptotic cells at 2, 4, and 10 h increased slightly over untreated mice but remained 10-fold lower than the wild-types at all time points. Similar results were also seen in the T-cell–containing white pulp of the spleen (results not shown). Atm heterozygous mice had an intermediate level of apoptosis at all time points in both thymus and spleen, indicating an Atm gene dosage effect on apoptotic sensitivity in lymphoid cells. p53 was induced by IR in all genotypes. The number of cells that were positive for nuclear p53 was comparable between genotypes (Fig. 1B), but the intensity of staining was lower in the Atm nulls (Fig. 1C and E). As another measure of p53 activity, we stained for the cyclin-dependent kinase inhibitor p21/waf1, which is induced in a p53-dependent manner. In both the cortex and medulla of irradiated Atm^–/– thymus, the number of p21-positive cells was 2- to 3-fold less than wild-type thymus (data not shown). The reduced intensity of p53, fewer p21-positive cells, and fewer apoptotic cells in irradiated Atm-null mice indicate that Atm plays a key role in regulating the p53 pathway in lymphoid tissue.

View larger version (59K): [in this window] [in a new window] [Download PPT slide]   FIGURE 1. Apoptosis and p53 induction in thymus from irradiated Atm-null, heterozygous, and wild-type mice. Numbers of apoptotic cells (A) and p53-positive cells (B) in thymic cortex of Atm+/+, Atm+/–, and Atm–/– mice at 0, 2, 4, and 10 h after 4 Gy IR. C. H&E and p53 staining in thymic cortex 4 h after 4 Gy IR. D. H&E, TUNEL, and caspase-3 staining in thymic cortex of Atm+/+, Atm+/–, and Atm–/– mice 10 h after 10 Gy IR. E. Western blot analysis of p53 protein from irradiated thymus 4 h after 4 Gy IR. Lamin C is used a loading control.

Apoptotic Response in Brain
In agreement with results of Herzog et al. (25), IR-induced apoptosis was impaired in the central nervous system of Atm-null mice. Within the dentate gyrus of the brain 4 h after 4 Gy IR, Atm-null mice averaged 0.2 apoptotic cells per 40x field compared with 1.8 per field in wild-type controls. There were also fewer Purkinje cells present in the Atm nulls compared with the wild-type (data not shown).

DNA Damage Response in Small Intestine
Epithelial cells within the stem cell compartment at the base of the crypt of the small intestine undergo p53-dependent apoptosis in response to IR (30). At 2 h after 4 Gy IR, apoptosis in Atm-null mice increased slightly from 0.2 in untreated to 0.7 cells per crypt, whereas wild-types had increased from 0.3 in untreated to almost 3 cells per crypt (Fig. 2A ). The Atm heterozygotes had an intermediate level of apoptosis, 1.6 cells per crypt at this time point. However, by 4 and 10 h, the numbers of apoptotic cells in the small intestine of the Atm-null mice were equivalent to wild-type. This short delay in apoptosis in Atm-deficient mice was more pronounced in younger mice. Three-week-old Atm^–/– mice irradiated with 4 Gy and examined 4 h later had 50% fewer apoptotic bodies (1.3 ± 0.2 cells per crypt, n = 5 mice) relative to Atm^+/+ mice (2.4 ± 0.2 cells per crypt, n = 5). In adult mice, the number of cells that stained positive for nuclear p53 was similar for all genotypes at all time points (Fig. 2B and C). Although p53 was clearly induced in crypt cells of Atm^–/– mice, it was possible that it was inactive (e.g., due to aberrant phosphorylation), and the apoptosis observed was p53 independent. To check for this possibility, we irradiated Atm^–/–p53^–/– compound mutant mice. Results show a nearly complete lack of radiation-induced apoptosis in intestinal crypt cells (Fig. 2A), similar to that seen in untreated mice or irradiated p53^–/– mice (30).

View larger version (55K): [in this window] [in a new window] [Download PPT slide]   FIGURE 2. Apoptosis and p53 induction in small intestine from irradiated Atm-null, heterozygous, and wild-type mice. Numbers of apoptotic cells (A) and p53-positive cells (B) in small intestine crypts of Atm+/+, Atm+/–, and Atm–/– mice at 0, 2, 4, and 10 h after 4 Gy IR. Striped column, apoptotic cells in Atm–/–p53–/– mice (n = 4) at 4 h. C. H&E, phospho-H2AX, phospho-Chk2, p21, BrdUrd, and histone H3 staining in small intestine crypts 2 h after 4 Gy IR. Arrows point to apoptotic cells. BrdUrd-positive cells (D) and mitotic cells (E) at 0, 2, and 4 h after 4 Gy IR.

Cell culture studies have shown that Atm participates in the G₂-M checkpoint (31). Atm-mutant cells irradiated in G₂ fail to arrest in G₂ and continue into mitosis. This arrest defect is attributed to impaired activation of the Chk2, Cdc25C, and Cdc2 checkpoint pathway (5). To determine if Atm regulates the G₂-M checkpoint in cells in their native environment, we quantified mitotic figures and staining for phospho-histone H3, a mitotic marker, at several times after whole-body IR. The mitotic index within the small intestinal crypts was similar between untreated wild-type and Atm-null mice. By 2 h after IR, mitotic figures were reduced to nearly zero in wild-type mice, indicative of an efficient G₂ arrest, but mitotic index remained high in the Atm nulls (Fig. 2E). Histone H3 staining confirmed this difference: the number of H3-positive cells decreased 35-fold in irradiated wild-type mice but only 3-fold in Atm-null mice (Fig. 2C). By 4 h, mitotic activity was nearly undetectable in both genotypes.

To further investigate the mechanism of this delay in cellular response to damage, we examined two proteins known to be phosphorylated by Atm: H2AX, a marker for DNA double-strand breaks, and Chk2, a regulator of the G₂-M checkpoint. Although radiation resulted in increased phospho-Chk2 in Atm-null mice, the number of phospho-Chk2–positive cells was reduced compared with wild-type mice (Fig. 2C). The increase in phospho-H2AX staining after IR was also diminished in Atm-null mice compared with wild-types (Fig. 2C). The fact that the DNA damage response at 2 h after IR was impaired in Atm-deficient mice, as shown by reduced phospho-Chk2 and phospho-H2AX staining, fewer apoptotic cells, and impaired G₂-M arrest, but was similar to wild-type by 4 h, suggests that Atm participates in the immediate early response to damage. Backup pathways can rapidly and efficiently compensate for the loss of Atm in this tissue. This is in contrast to thymus and spleen, which showed impaired apoptosis in Atm-null mice at all time points.

DNA Damage Response in Epidermis
As in the intestinal crypt, p53 is required for IR-induced apoptosis within epithelial cells of the hair follicle (32). p53 was induced by IR in basal epithelial and hair follicle cells in both wild-type and Atm-deficient mice with similar kinetics (Fig. 3 ). We also tested younger animals, at an age of 3 weeks, when there is a higher population of proliferating cells in the skin. Again, the numbers of apoptotic cells were similar in all genotypes (data not shown). In over 20 animals of each genotype examined, the number of p53-positive cells and apoptotic bodies was similar between Atm^–/– and wild-type controls regardless of age, radiation dose, or time point. This radiation-induced apoptosis in hair follicle cells is also p53 dependent as it was not observed in Atm^–/–p53^–/– animals.

View larger version (71K): [in this window] [in a new window] [Download PPT slide]   FIGURE 3. Apoptosis and p53 induction in epidermis from irradiated Atm-null, heterozygous, and wild-type mice. Numbers of apoptotic cells (A) and p53-positive cells (B) in skin of Atm+/+, Atm+/–, and Atm–/– mice at 0, 2, 4, and 10 h after 4 Gy IR. C. H&E and p53 staining in hair follicles 4 h after 4 Gy IR. Arrows point to p53-positive and apoptotic cells.

	Discussion

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Using a defined IR dose and a standard method of assessing p53 staining and apoptosis, we show marked tissue-specific differences in the role of Atm in the DNA damage response. In thymus, spleen, and brain, Atm was required for IR-induced apoptosis, whereas in epithelial cells of the gastrointestinal tract and epidermis, after a slight delay, Atm was largely dispensable. This provides an explanation for discrepant results obtained in different cell culture models for p53 regulation by Atm. More importantly, the variable requirements for Atm in regulating p53 imply that other pathways are operative and the importance of these pathways varies in a development- and tissue-specific manner.

Reduced apoptosis in Atm-deficient thymus was not restored by high doses or later time points, indicating that DNA damage-induced apoptosis is highly dependent on Atm in this tissue and backup pathways do not come into play with increasing damage or time after damage. Other studies showed that Atm^–/– thymocytes are resistant to IR-induced apoptosis (22, 23), whereas two reported no defect in p53-mediated thymic apoptosis (24, 25). The latter two studies used TUNEL to identify apoptotic cells. In our hands, the TUNEL assay did not detect differences in apoptosis between Atm genotypes, whereas active caspase-3 antibodies and morphology-based microscopy did, suggesting that TUNEL may be misleading in certain tissues or genetic contexts. The intensity of staining for p53 was reduced in irradiated Atm-null thymus, although the number of positive cells was similar, further evidence for an impaired p53 response. This also suggests that reduced p53 in Atm-deficient cells seen by Western blot analysis is likely due to reduced p53 per cell rather than fewer cells that induce p53. The observation that Atm^+/– mice showed intermediate levels of apoptotic sensitivity establishes that Atm is haploinsufficient for DNA damage-induced apoptosis. Xu and Baltimore (22) also reported impaired apoptosis in Atm^+/– thymocytes, further highlighting the sensitivity of these cells to the Atm pathway.

In contrast to the requisite role of Atm in the DNA damage response in thymus, Atm was largely dispensable in epithelial cells of the intestine and skin. The only difference noted was a 2-h delay in apoptosis and G₂ cell cycle arrest in the crypt cells of the intestine. Reduced phospo-Chk2, phospho-H2AX, and p21 at this time point are consistent with impaired Atm signaling. This shows that Atm functions in the immediate response to DNA damage through at least two pathways: p53-mediated apoptosis and Chk2-mediated G₂ arrest. Both apoptosis and cell cycle arrest reached wild-type levels by 4 h, indicating that other proteins compensate for the loss of Atm in this tissue. Likely possibilities are other members of the phosphatidylinositol 3-kinase–like family, such as ATR and DNA-PK. DNA-PK, like Atm, is able to phosphorylate p53 (33), Chk2 (34), and H2AX (35). We previously reported that p53 induction and apoptosis are not impaired in DNA-PK–mutant mice, indicating that neither DNA-PK nor Atm on their own is required for apoptosis (36). We attempted to generate Atm/DNA-PK double-mutant mice to determine if Atm and DNA-PK redundantly regulated p53 and apoptosis, but early embryonic lethality of the double mutants precluded this analysis (37).

The molecular basis for the radiosensitivity of Atm-deficient cells or mice is poorly understood (3). To examine the role of p53, Westphal et al. (23) generated Atm/p53 compound mutant mice and showed that these mice were as radiosensitive as Atm mice. This was attributed to loss of intestinal crypts and failure to regenerate intestinal epithelia. As Atm/p53 compound mutant crypt cells are resistant to IR-induced apoptosis at 4 h (Fig. 1A), this uncouples the early wave of apoptosis from radiosensitivity. Alternative mechanisms leading to Atm radiosensitivity could be inefficient G₂-M arrest, leading to chromosome breaks and mitotic catastrophe or damage to stem cells (38, 39). IR-induced p53 and apoptosis in the epidermis were similar between Atm genotypes at all time points. This was surprising, as skin-derived cells from patients with ataxia-telangiectasia are highly radiosensitive (40). Increased phosphorylated p53 and H2AX in irradiated wild-type and Atm-deficient mouse skin have been reported (41). This again suggests that early markers of DNA damage response or apoptosis do not necessarily predict radiosensitivity of the tissue.

The basis for the narrow tumor predisposition of Atm-deficient mice is unclear. Atm-null mice have only been reported to show increased susceptibility to lymphoid tumors and a modest increase in squamous cell tumors in a Myc transgenic model (42). Two possibilities are as follows: a unique role of Atm in lymphoid lineages or a specific lack of redundant pathways to Atm in lymphoid tissue, which would expose the vulnerability of Atm loss. Liao and Van Dyke (43) concluded that there were two different mechanisms of tumor suppression by Atm and p53. Lymphoma suppression in Atm-null mice depended on V(D)J recombination, whereas lymphomas from p53-null mice arose independent of V(D)J recombination. Atm has recently been directly implicated in repair of V(D)J breaks during antigen receptor rearrangement, which could result in increased oncogenic translocations (44). Consistent with this idea, translocations involving the antigen receptors are frequently seen in lymphoid tumors from both ataxia-telangiectasia patients (45) and Atm^–/– mice (22, 24). This tissue-specific role of Atm, together with its requisite role in regulating apoptosis in lymphocytes, could explain the particular sensitivity of Atm-deficient mice and ataxia-telangiectasia patients to lymphoid tumors. More generally, the observation that there are marked tissue-specific differences in the DNA damage pathway implies that the role of DNA damage as a barrier to cancer (26, 27) is also likely to be tumor type specific.

	Materials and Methods

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Mice
129/SvEv Atm^(ins5790neo) knockout mice were obtained from A. Wynshaw-Boris (19). Atm heterozygous breeder pairs were set up to generate Atm-null, heterozygous, and wild-type littermates. To generate Atm p53 double-null mice, Atm heterozygous mice were crossed to p53 knockout mice (46), and resulting double heterozygous offspring set up as breeder pairs. Genotyping was done using previously published methods for p53 and Atm (47, 48). At 7 to 10 weeks of age, mice were given either 4 or 10 Gy of whole-body IR using a ⁶⁰Co source. Mice were then sacrificed at 2, 4, or 10 h after IR. Some mice were injected with BrdUrd (100 mg/kg, i.p.) 1 h before sacrifice. Unirradiated age-matched controls were also sacrificed for tissue examination.

Histology
Sections of skin, thymus, spleen, brain, and small intestine were snap frozen or fixed for 4 h in normal buffered formalin and then processed to paraffin. Sections (4 µm) were cut, deparaffinized, and stained for either H&E, p53 (CM5, Novocastra), BrdUrd (DAKO), cleaved caspase-3 (Asp¹⁷⁵; Cell Signaling Technology), p21 (BD PharMingen), phospho-Chk2 (Abcam), phospho-histone H2AX (Ser¹³⁹; Cell Signaling Technology), or phospho-histone H3 (Ser¹⁰; Cell Signaling Technology). Staining for p53, caspase-3, p21, phospho-Chk2, and histone H3 was done using a three-step streptavidin technique. Sections were rehydrated and treated with high heat antigen retrieval using a 10 mmol/L citrate buffer (pH 6) and then stained with primary antibody. Sections stained for BrdUrd were rehydrated and treated with HCl and trypsin followed by the primary antibody. For all stains, a biotinylated secondary antibody was used and then a streptavidin-horseradish peroxidase conjugate (DAKO). Some tissues were also cut for TUNEL. TUNEL was done according to Gavrieli et al. (49). Briefly, deparaffinized and rehydrated sections were treated with proteinase K and then incubated with terminal deoxynucleotidyl transferase and biotin-14-dATP followed by streptavidin-horseradish peroxidase. All sections were developed in 3,3'-diaminobenzidine/NiCl and counterstained with methyl green.

Immunoblotting
Nuclear protein extracts were prepared as described (50) with modifications. Buffers A and C both contained 1 mmol/L DTT, 0.4 mg/mL Pefabloc, 25 mg/mL aprotinin, and 10 mg/mL leupeptin (Roche) to inhibit proteases. Protein concentrations were measured using the Bradford assay (Bio-Rad) and equal loading was confirmed by Ponceau S (Sigma) staining of the nitrocellulose membrane. Primary antibody CM5 p53 antibody was used and then developed using a SuperSignal West Femto kit (Pierce). Membrane was stripped using Chemicon stripping buffer (Fisher Scientific) and reprobed using lamin A/C antibody (Cell Signaling Technology).

Quantification
Numbers of apoptotic figures, mitotic figures, or stained positive cells were counted in 10 100x fields in the thymic cortex, in 10 40x fields in the small intestine crypts, or in 10 hair follicles in the skin. There is an average of 8 crypts per field so 10 fields = 80 crypts. Three to eight mice were used per treatment group. Results were expressed as positive cells per field, positive cells per crypt, or positive cells per follicle. Sections were examined blind to the genotype to avoid bias. Significance between groups was determined using the unpaired t test (GraphPad Software).

	Acknowledgements

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
We thank Tony Wynshaw-Boris for generously providing Atm knockout mice and Kyung-Hoon Kim and Ann Marie Shen for assistance.

	Notes

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Grant support: NIH R01 research grants CA 70414 and CA 099517 (C.J. Kemp) and NIEHS research grant U01 ES11045 (C.J. Kemp).

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.

Received 5/19/07; revised 7/16/07; accepted 7/26/07.

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