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Molecular Cancer Research 5, 705-711, July 1, 2007. doi: 10.1158/1541-7786.MCR-07-0058
© 2007 American Association for Cancer Research
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Cell Cycle, Cell Death, and Senescence

Atm Deficiency Affects Both Apoptosis and Proliferation to Augment Myc-Induced Lymphomagenesis

Kirsteen H. Maclean1,2, Michael B. Kastan2 and John L. Cleveland1,3

Departments of 1 Biochemistry and 2 Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee and 3 Department of Cancer Biology, The Scripps Research Institute-Florida, Jupiter, Florida

Requests for reprints: John L. Cleveland, Department of Cancer Biology, The Scripps Research Institute-Florida, 5353 Parkside Drive, Jupiter, FL 33458. Phone: 561-799-8808. E-mail: jcleve{at}scripps.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Myc oncoproteins are commonly activated in malignancies and are sufficient to provoke many types of cancer. However, the critical mechanisms by which Myc contributes to malignant transformation are not clear. DNA damage seems to be an important initiating event in tumorigenesis. Here, we show that although Myc does not directly induce double-stranded DNA breaks, it does augment activation of the Atm/p53 DNA damage response pathway, suggesting that Atm may function as a guardian against Myc-induced transformation. Indeed, we show that Atm loss augments Myc-induced lymphomagenesis and impairs Myc-induced apoptosis, which normally harnesses Myc-driven tumorigenesis. Surprisingly, Atm loss also augments the proliferative response induced by Myc, and this augmentation is associated with enhanced suppression of the expression of the cyclin-dependent kinase inhibitor p27Kip1. Therefore, regulation of cell proliferation and p27Kip1 seems to be a contributing mechanism by which Atm holds tumor formation in check. (Mol Cancer Res 2007;5(7):705–11)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Resolving the precise mechanisms by which oncogenes such as Myc promote tumorigenesis is a major thrust of oncology research. At one level, this involves a hyperproliferative response provoked by Myc, because when Myc is overexpressed, it accelerates the rates of cell proliferation (1, 2) and increases the numbers of cells in cycle in vivo (3-5). This enhanced proliferative response is held in check by the universal cyclin-dependent kinase inhibitor p27Kip1 and by the Arf-p53 pathway, which also mediates Myc-induced apoptosis; bypass of these inhibitory steps are common denominators of cancer (5-11). However, Myc overexpression also sensitizes cells to agents that induce DNA damage and leads to genomic instability (12-16), suggesting that alterations in the DNA damage and/or DNA repair pathways might contribute to mutations augmented by Myc activation.

A key arbiter of the DNA damage pathway is the ataxia-telangiectasia mutated (ATM) gene, a member of the phosphatidylinositol-3-OH kinase superfamily. ATM is activated by DNA damage and phosphorylates key substrates that arrest the cell cycle in the G1 (e.g., p53), S, and G2-M phases of the cell cycle (17). Ataxia-telangiectasia patients with loss-of-function mutations in ATM suffer from cerebellar degeneration causing ataxia and are prone to development of malignancies, particularly lymphomas (18). Furthermore, sporadic, generally missense, inactivating mutations of ATM, with loss of the wild-type ATM allele, have been reported in mantle cell lymphoma, T-cell prolymphocytic leukemia, and B-cell chronic lymphocytic leukemia (19). Finally, Atm deficiency in mice results in the development of thymic lymphoma (20, 21). Collectively, these data all indicate that ATM functions as a bona fide tumor suppressor.

Atm phosphorylates and activates p53 (17) and p53 mediates Myc-induced apoptosis (7, 8, 22-24). Further, Myc has been suggested to directly provoke DNA damage (14, 25) and sensitize cells to irradiation (15), suggesting potential interplay between Myc and Atm in tumorigenesis. Indeed, in epithelial cells, Atm deficiency has been shown to cooperate with Myc in the development of oral and skin tumors, and in this scenario Atm loss impairs Myc-induced apoptosis (25). Here, we report that although Myc does not directly induce DNA strand breaks in proliferating cells ex vivo or in B cells in vivo, it does augment the Atm/p53 DNA damage pathway. Importantly, the Atm tumor suppressor is shown to harness Myc-induced lymphomagenesis at two levels: by impairing Myc-induced apoptosis and by augmenting its proliferative response, with the latter linking Atm to the control of p27Kip1 cyclin-dependent kinase inhibitor.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Myc Augments the DNA Damage Response to Irradiation
To initially assess the possible interplay between Myc, Atm, and the DNA damage response, primary early-passage human diploid foreskin fibroblasts were transduced with a control MSCV-IRES-puro retrovirus, which expresses the gene for puromycin resistance (puro) in cis from an internal ribosome entry site (IRES), or with the MSCV-Myc-ERTAM-IRES-puro retrovirus (7) that additionally encodes a chimeric form of c-Myc fused to the estrogen binding domain of an estrogen receptor (ER) modified to selectively bind to the estrogen receptor agonist tamoxifen (TAM). In the presence of tamoxifen, the Myc-ERTAM fusion protein relocalizes to the nucleus and activates Myc transcriptional programs (26). Puromycin-resistant cells were expanded in culture, treated with tamoxifen to activate Myc-ERTAM, and were then exposed to 5-Gy ionizing radiation. The effects of Myc on ATM activation were then monitored by evaluating Ser1981 phosphorylation of ATM, a marker of ATM activation (27), and by evaluating the levels of p53 protein and damage-dependent p53 phosphorylation on Ser15 (28, 29).

As expected, ionizing radiation induced the Ser1981 phosphorylation of ATM and the Ser15 phosphorylation of p53 in control, vector-only–expressing fibroblast cells (Fig. 1A ). By contrast, activation of Myc alone did not induce ATM phosphorylation, but it was sufficient to induce p53 and Ser15 p53 phosphorylation (Fig. 1A). The expression of the BH3-only p53 transcription target Puma, a proapoptotic Bcl-2 family member (30-32), was also elevated in Myc-ERTAM–expressing human foreskin fibroblasts (Fig. 1A), even without activation of the Myc-ERTAM transgene, which is likely due to its leaky activation. Notably, however, Myc activation did augment Ser1981 ATM phosphorylation in response to ionizing radiation (Fig. 1A); therefore, Myc overexpression does not seem to induce direct DNA damage per se in proliferating human foreskin fibroblasts, but rather enhances activation of the ATM-p53 pathway following DNA damage.


Figure 1
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  		FIGURE 1. Myc augments the DNA damage signaling pathway in normal human diploid foreskin fibroblasts. A. Primary early-passage cultures of human foreskin fibroblasts were transduced with the MSCV-IRES-puro retrovirus or with the MSCV-Myc-ERTAM-IRES-puro retrovirus, and puromycin-resistant pools of cells were then left untreated (Unt.) or treated with 1 µmol/L 4-hydroxy tamoxifen (4HT) for 24 h. Half of these cells were then treated with 5 Gy of ionizing radiation ({gamma}-IR). After 4 h, samples were then evaluated for their levels of phospho-Ser1981 ATM, p53, phospho-Ser15 p53, Puma, and ß-actin by immunoblotting. B. Vector-only– (Puro) or Myc-ERTAM–expressing human foreskin fibroblasts (Myc-ERTAM) were treated with 4-hydroxy tamoxifen (1 µmol/L) for 24 h, followed by 5-Gy ionizing radiation, and harvested after 4 h. Following fixation and blocking procedures, double-strand breaks in DNA were detected by incubating with anti–Ser139-H2AX antibody (p-H2AX). Cells were then stained with a Cy3 antirabbit secondary antibody. DNA was visualized with 4',6-diamidino-2-phenylindole (DNA). Representative of three separate experiments.

 
Damage that provokes double-strand breaks in DNA triggers Ser139 phosphorylation of H2AX, which then accumulates at discrete foci in the nuclei of damaged cells (33). As expected, exposure of both vector-only– and Myc-ERTAM–overexpressing cells to ionizing radiation led to the formation of discrete H2AX foci (Fig. 1B). However, Myc activation alone, which fails to induce ATM phosphorylation (Fig. 1A), also failed to induce H2AX foci (Fig. 1B). Therefore, Myc overexpression alone can provoke some (e.g., Ser15 phosphorylation of p53), but not all, hallmarks of the DNA damage response.

Myc Augments Ser15 p53 Phosphorylation In vivo
Precancerous B cells of Eµ-Myc transgenic mice, a mouse model of human Burkitt lymphoma that bears MYC/Immunoglobulin translocations (34), are exquisitely sensitive to ionizing radiation (15), suggesting that some aspects of the DNA damage pathway might also be activated by Myc overexpression in vivo. Indeed, compared with expression in B cells from wild-type littermate mice, there were marked increases in the steady-state levels of p53 and Ser15 phosphorylated p53 in both spleen- and bone marrow–derived B220+ B cells from precancerous Eµ-Myc transgenic mice. Increased levels of Puma were also detected in Eµ-Myc splenic B cells (Fig. 2A ). However, similar to the effects observed during Myc activation in human fibroblasts (Fig. 1B), H2AX foci were not detected in B220+ B cells isolated from spleen of precancerous Eµ-Myc transgenic mice, and only a few foci-positive cells were detected in bone marrow–derived Eµ-Myc B220+ B cells (Fig. 2B).


Figure 2
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  		FIGURE 2. Myc augments the DNA damage signaling pathway in vivo. A. B220+ spleen- and bone marrow–derived B cells from 4-wk-old Eµ-Myc transgenic mice and their wild-type (Wt) littermates were evaluated for their levels of phospho-Ser15 p53, p53, and Puma by Western blot. B. B cells derived from either the bone marrow or spleen of wild-type or Eµ-Myc mice were analyzed for the presence of double-strand breaks in DNA by incubating with anti–Ser139-H2AX antibody (p-H2AX). Cells were then stained with a Cy3 antirabbit secondary antibody. DNA was visualized with 4',6-diamidino-2-phenylindole (DNA). Representative of three separate experiments.

 
Atm Functions as a Guardian against Myc-Induced Lymphomagenesis
Ataxia-telangiectasia patients, who have loss of function mutations in ATM, are highly prone to develop lymphoid leukemia and lymphoma during childhood (35), and Atm-deficient mice spontaneously develop malignant thymic lymphomas by 4 to 6 months of age (20, 21). Because evidence of activation of DNA damage signaling pathways has been detected in precancerous cells of mice and men (36, 37), we reasoned that loss of ATM might accelerate tumorigenesis in the face of an oncogenic insult. To test this hypothesis, Atm+/– mice were crossed with Eµ-Myc transgenic mice, which develop clonal, lethal pre–B or B-cell lympholeukemia by 3 to 6 months of age (34). Eµ-Myc;Atm+/– F1 mice were then mated with Atm+/– mice, and Eµ-Myc;Atm+/+, Eµ-Myc;Atm+/–, Eµ-Myc;Atm–/–, and Atm–/– littermates were followed for their course of disease. As expected, nontransgenic Atm–/– mice died of thymic lymphoma with a median survival time of 160 days, whereas Eµ-Myc;Atm+/+ mice died of pre-B and B-cell lymphoma with a median survival of 120 days (Fig. 3 ). Notably, although no effects of Atm haploinsufficiency were evident (median survival, 127 days), all Eµ-Myc;Atm–/– mice developed aggressive, rapid-onset pre-B and B-cell lympholeukemia (mean survival, 69 days; Fig. 3). Therefore, Atm indeed serves as a guardian against Myc-induced lymphomagenesis.


Figure 3
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  		FIGURE 3. Atm functions as a guardian against Myc-induced lymphomagenesis. Eµ-Myc transgenic mice (on a C57BL/6 background) were crossed with C57BL/6 Atm+/– mice. F1 Eµ-Myc;Atm+/– mice were then mated with Atm+/– mice, and Eµ-Myc;Atm+/+ (n = 40; black line), Eµ-Myc;Atm+/– (n = 66; red line), Eµ-Myc;Atm–/– (n = 13; blue line), and Atm–/– (n = 14; green line) littermates were followed for their course of lymphoma development. Median survival time was 120 d for Eµ-Myc;Atm+/+, 127 d for Eµ-Myc;Atm+/–, and 69 d for Eµ-Myc;Atm–/– (P = 0.003) transgenic mice. Median survival time of Atm–/– littermates was 160 d. Lymphomas that arose in all Eµ-Myc transgenics were pre-B or immature B-cell lymphoma, whereas only thymic lymphoma arose in Atm–/– mice.

 
Loss of Atm Impairs Myc Apoptotic Responses
Myc accelerates the rate of cell proliferation and augments apoptosis in precancerous B cells of Eµ-Myc transgenic mice (3-5, 38), and thus loss of cell cycle regulators such as p27Kip1 or of apoptotic regulators such as Arf or p53 dramatically increases the rate of lymphoma development (7-9). The acceleration of pre-B and B-cell lymphoma development in Eµ-Myc;Atm–/– transgenic mice could result from effects of Atm deficiency on Myc-induced apoptosis, Myc-induced proliferation, or both. To assess the apoptotic response, we evaluated B cells derived from precancerous (4-week-old) Eµ-Myc;Atm+/+, Eµ-Myc;Atm–/–, Atm–/–, and wild-type mice cultured in interleukin-7–containing medium on S17 stromal cells (15), which selectively allows for the outgrowth of B220+ B cells from bone marrow (39). Atm deficiency alone had little effect on spontaneous apoptosis in primary B-cell cultures, whereas rates of spontaneous apoptosis of Eµ-Myc;Atm+/+ B cells were, as expected (7), quite high (Fig. 4A ). Notably, rates of spontaneous apoptosis of Eµ-Myc;Atm–/– were comparable with those of wild-type B cells (Fig. 4A), indicating that Atm deficiency can effectively inhibit the Myc-induced apoptotic response.


Figure 4
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  		FIGURE 4. Atm loss impairs Myc apoptotic pathways. A. Primary B cells were grown on S17 stroma and in media supplemented with interleukin-7 from 4-week-old nontransgenic (wild-type), Eµ-Myc, Atm–/–, and Eµ-Myc;Atm–/– mice. The percent of spontaneous apoptosis was determined by Annexin V-FITC. Columns, mean of three independent B-cell cultures for the indicated mice; bars, SD. B. Lysates were prepared from bone marrow– and spleen-derived B cells from 4-week-old nontransgenic (wild-type), Eµ-Myc, Atm–/–, and Eµ-Myc;Atm–/– mice and were evaluated for their levels of p53, p19Arf, Puma, Bcl-XL, Bcl-2, p27Kip1, and ß-actin by Western blot.

 
Myc-induced apoptosis in primary B cells is governed by activation of the Arf-p53 pathway and also through suppressing the expression of antiapoptotic Bcl-2 family members such as Bcl-XL and Bcl-2 (7, 15, 40). We therefore directly compared the expression of these regulators and of Puma in magnetic-activated cell sorted B220+ B cells from the spleens or bone marrows of Eµ-Myc;Atm+/+, Eµ-Myc;Atm–/–, Atm–/–, and wild-type weanling-aged (4-week-old) littermates. As expected, Eµ-Myc;Atm+/+ B cells expressed higher levels of p19Arf, p53, and Puma and reduced levels of Bcl-XL and Bcl-2, compared with the B220+ B cells from the bone marrows or spleens of nontransgenic Atm–/– or wild-type littermates (Fig. 4B). Atm deficiency affected both arms of Myc's apoptotic response because Eµ-Myc;Atm–/– B cells expressed both reduced levels of p53, p19Arf, and Puma and increased levels of Bcl-XL (and of Bcl-2 in splenic B cells) compared with wild-type Eµ-Myc transgenics (Fig. 4B).

Atm Deficiency Augments Myc Proliferative Response
Myc accelerates the rate of cell proliferation through its ability to provoke degradation of the cyclin-dependent kinase inhibitor p27Kip1 (5, 6). Loss of p27Kip1 markedly accelerates the rate of Myc-induced lymphomagenesis in the Eµ-Myc mouse model (9), whereas E2f1 or Cks1 deficiency disables Myc's ability to suppress p27Kip1 and thus impairs lymphomagenesis (3, 5). To assess the potential effects of Atm deficiency on Myc-induced proliferation, weanling wild-type Eµ-Myc and Eµ-Myc;Atm–/– littermates were injected with bromodeoxyuridine (BrdUrd) and, after 12 h, the S-phase indices of their bone marrow and splenic B220+ B cells were determined by flow cytometry. As expected (3-5), the proliferative indices of both immature (bone marrow) and mature (spleen) B cells were augmented in Eµ-Myc B cells (Fig. 5 ). Surprisingly, this proliferative response was even higher in B cells from Eµ-Myc;Atm–/– mice (Fig. 5). Finally, the markedly high rates of proliferation of Eµ-Myc;Atm–/– cells were associated with drastically reduced levels of the p27Kip1 cyclin-dependent kinase inhibitor (Fig. 4D). Therefore, Atm seems to hold Myc-induced transformation of B cells in check by compromising both the apoptotic and proliferative responses induced by Myc.


Figure 5
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  		FIGURE 5. Atm loss augments Myc-induced proliferation in vivo. Four-week-old nontransgenic (wild-type), Eµ-Myc, and Eµ-Myc;Atm–/– littermates were injected with BrdUrd (BrdU) and, after 12 h, the percentage of B220+ B cells (derived from both bone marrow and spleen) in S phase was determined by fluorescence-activated cell sorting. Columns, mean of three independent experiments from the indicated mice; bars, SD.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
Myc has been recognized to trigger genomic instability for a number of years (12, 13, 16), yet precisely how this occurs has not been resolved. Under some scenarios, this phenomenon has been linked to the ability of Myc to directly induce double-strand breaks in DNA and {gamma}-H2AX foci (41), which may or may not occur through augmenting levels of reactive oxygen species (14, 41) or via direct activation of Atm (42). However, in other settings, Myc does not induce reactive oxygen species (43) and, as shown here, Myc overexpression in normal proliferating human fibroblasts and B cells in vivo does not measurably induce ATM activation or {gamma}-H2AX foci. Nevertheless, Myc still clearly sensitizes cells to apoptosis following DNA damage (15) and augments activation of ATM in response to {gamma}-ionizing radiation (Fig. 1A). Further, loss of Atm, a key regulator of the DNA damage response, impairs Myc-induced apoptosis and also augments Myc proliferative response. Thus, Atm loss markedly accelerates lymphoma development in Eµ-Myc transgenic mice, a model of human Burkitt lymphoma (34).

Several previous observations predict that disabling the Atm-p53 pathway would enhance Myc-induced tumorigenesis by specifically impairing Myc apoptotic response: (a) loss of p53 function augments Myc-induced transformation by impairing its apoptotic response (7, 8, 23, 24); (b) blocking apoptosis by modulation of Bcl-2 family members also augments Myc-induced tumorigenesis (32, 40, 44-47); (c) ATM is a protein kinase that signals to p53 and is required for optimal p53 induction after ionizing radiation (17); (d) loss of Atm leads to decreased apoptosis of immature thymocytes and brain cells in mice exposed to irradiation (48); and (e) inactivation of Atm in a transgenic mouse model overexpressing Myc in squamous epithelial tissues leads to decreased apoptosis and rapid onset of tumor development (25). Similar to studies of Myc-driven tumorigenesis in keratinocytes (25) and to very recent findings by Shreeram et al. (49) evaluating the effects of the Atm-p53 pathway downstream of the Wip1 phosphatase in Eµ-Myc mice, we also observed that Atm deficiency disables Myc apoptotic response. Notably, here we have shown that this is specifically associated with reductions in the degree of activation of the Arf-p53 pathway in the precancerous state as well as to unexpected effects of Atm deficiency on the expression of Bcl-2 and Bcl-XL in these at-risk cells (Fig. 4B). Precisely how Atm deficiency would dampen these two apoptotic pathways is not clear but could conceivably be due to the effects of Atm loss on other checkpoints that have ancillary effects on these pathways. Finally, these findings suggest that alterations which disable the Arf-p53 pathway and often accompany lymphoma development in Eµ-Myc mice would be more infrequent in an Atm-null context, as suggested by others (49), but this issue will have to be addressed using large cohorts of these lymphomas.

A surprising finding of our studies was that Atm deficiency also augments Myc-driven proliferation, which is also rate limiting for lymphoma development in Eµ-Myc mice (3, 5, 9). Myc accelerates the rate of cell proliferation (1, 2), at least in part, through its ability to provoke degradation of the cyclin-dependent kinase inhibitor p27Kip1 (5, 6), and the combined effect of Myc overexpression and Atm deficiency effectively abolishes p27Kip1 expression and drives high rates of cell proliferation. Precisely how Atm deficiency affects the Myc-to-p27Kip1 pathway is also not clear, but, at one level, this might involve the effects of Atm loss on E2f1 expression and/or activity because proper thresholds of E2f1 are necessary for Myc to suppress p27Kip1 expression in Eµ-Myc B cells (3). The dual tumor suppressor mechanism (apoptosis and growth arrest) suggested here for ATM is similar to the scenario for p53, where both its proapoptotic (8, 50) and antiproliferative (51) effects have been shown to contribute to its tumor-suppressive properties.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Mice and Cell Culture
Atm+/– mice on a C57BL/6;Svj129 mixed background (kindly provided by Dr. Peter McKinnon; ref. 48) were crossed with Eµ-Myc transgenic mice (C57BL/6 background; ref. 34) to generate F1 Eµ-Myc;Atm+/– mice. These mice were then crossed to Atm+/– mice to generate Eµ-Myc;Atm+/+, Eµ-Myc;Atm+/–, Eµ-Myc;Atm–/–, and Atm–/– offspring and these littermates were followed for their course of disease.

Primary bone marrow–derived pre-B-cell cultures were generated from 4- to 6-week-old wild-type, Eµ-Myc;Atm+/+, Eµ-Myc;Atm–/–, and Atm–/– littermates as previously described (40). B cells were maintained in culture on S17 stromal cells (S17 stroma kindly provided by Dr. Kenneth Dorshkind, University of California at Los Angeles, Los Angeles, CA; ref. 39) in medium containing interleukin-7 (10 units/mL) as described (40).

Normal human foreskin fibroblasts (American Type Culture Collection) were maintained in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin. Retroviral infections were done as previously described (7, 15).

Fluorescence-Activated Cell Sorting and Magnetic-Activated Cell Sorting of B Cells
To obtain bone marrow and splenic B cells, single-cell suspensions were prepared, followed by a red cell lysis using an ammonium chloride/potassium bicarbonate solution. Cell suspensions were then incubated with B220 microbeads and enriched by magnetic-activated cell sorting according to the manufacturer's instructions (Miltenyi Biotech). Rates of proliferation of B220+ cells were determined using a flow kit as described by the manufacturer (BD Biosciences PharMingen). Briefly, animals were injected i.p. with 100 µL of 10 mg/mL BrdUrd in sterile PBS. Animals were sacrificed 12 h postinjection and B220+ cells from bone marrow and spleen were harvested. For the BrdUrd proliferation assays, 1 x 106 cells were used. Following incubation, cells were washed, resuspended in PBS, and analyzed by fluorescence-activated cell sorting.

Apoptosis Assays
To assess the effects of Atm deficiency on Myc-induced apoptosis, bone marrows from individual 4-week-old wild-type, Eµ-Myc;Atm+/+, Eµ-Myc;Atm–/–, and Atm–/– littermates were cultured on S17 stroma in interleukin-7 medium as described (40). After 10 days in culture, the rates of spontaneous apoptosis of the pre-B cells in these cultures were determined by staining 5 x 105 cells with Annexin V-FITC antibody (Annexin V-Fluor Kit, Roche Applied Sciences) and propidium iodide as previously described (4). Following incubation, cells were washed, resuspended in PBS, and analyzed by fluorescence-activated cell sorting.

Protein Analyses
Protein extracts from magnetic-activated cell sorted B cells and human foreskin fibroblasts were prepared as previously described (7). Proteins (25 or 50 µg per lane) were electrophoretically separated on 4% to 12% SDS-PAGE gels (Invitrogen), transferred to membranes (Protran, Schleicher & Schuell), and blotted with antibodies specific for Puma, ß-actin (Sigma), p27Kip1, Bcl-XL, Bcl-2, p53, phospho-Ser15 p53 (Cell Signaling), p19Arf (kindly provided by Dr. Charles Sherr), and phospho-Ser1981 ATM (Abcam). Following incubation with primary antibodies, the blots were then incubated with appropriate antimouse or antirabbit immunoglobulin secondary antibodies (Amersham). Bound immunocomplexes were detected by enhanced chemiluminescence (Amersham) or ECL SuperSignal (Pierce).

Immunofluorescence Analyses
For immunofluorescence analysis of levels of Ser139 phosphorylated H2AX, cytospins of 5 x 104 B220+ B cells isolated from 4-week-old wild-type or Eµ-Myc transgenic mice, or from human fibroblasts (control or Myc-ERTAM-expressing) grown on glass coverslips, were prepared. Slides were fixed with 1:1 (v/v) methanol-acetone for 10 min at –20°C and were air dried. After blocking with 10% fetal bovine serum/PBS, slides were incubated for 1 h at room temperature with a polyclonal antibody specific for phospho-Ser139 histone {gamma}-H2AX [1:100 dilution in 1% fetal bovine serum/PBS (Trevigen)]. Following 10 washes in PBS, primary antibody binding was visualized with an antirabbit antibody conjugate incubated at room temperature for 30 min. Coverslips and slides were washed 10 times before being mounted with Fluoromount-G antifade reagent containing 4',6-diamidino-2-phenylindole (Southern Biotechnology) and were analyzed by confocal microscopy.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Charles Sherr and Martine Roussel (St. Jude Children's Research Hospital, Memphis, TN) for providing p19Arf antibody; Peter McKinnon (St. Jude Children's Research Hospital, Memphis, TN) for providing Atm+/– mice; Chunying Yang, Lottie Peppers, Elsie White, Diane Woods, Heather Briley, and Margaret Reis for outstanding technical assistance; and the Core Services of the Fluorescence-Activated Cell Sorting Facility, the Animal Research Center, and the Hartwell Center of St. Jude Children's Research Hospital.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Grant support: NIH grants CA76379 (J.L. Cleveland) and CA71387 (M.B. Kastan), the Cancer Center (CORE) support grant CA21765, and the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital.

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: The editor-in-chief of Molecular Cancer Research is a coauthor of this paper. In keeping with the AACR's Editorial Policy, a member of the AACR's Publications Committee had the paper reviewed independently of the journal's editorial process and made the decision whether to accept the paper.

Received 2/ 3/07; revised 4/19/07; accepted 4/25/07.


   References
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 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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