[Cancer Research 66, 2907-2912, March 15, 2006]
© 2006 American Association for Cancer Research
Defective p53 Response and Apoptosis Associated with an Ataxia-Telangiectasia–like Phenotype
Nuri Gueven1,
Olivier J. Becherel1,
Geoff Birrell1,
Philip Chen1,
Giannino DelSal3,
James P. Carney4,
Padraic Grattan-Smith5 and
Martin F. Lavin1,2
1 Queensland Institute of Medical Research; 2 Central Clinical School, University of Queensland, Brisbane, Queensland, Australia; 3 Laboratorio Nazionale CIB, Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Trieste, Italy; 4 Radiation Oncology Research Laboratory, Department of Radiation Oncology, University of Maryland School of Medicine, Baltimore, Maryland; and 5 Sydney Children's Hospital, Randwick, New South Wales, Australia
Requests for reprints: Martin F. Lavin, Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia. Phone: 61-7-33-62-0335; Fax: 61-7-33-62-0106; E-mail: martinL{at}qimr.edu.au.
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Abstract
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Ataxia-telangiectasia mutated (ATM), the protein defective in ataxia-telangiectasia, plays a central role in DNA damage response and signaling to cell cycle checkpoints. We describe here a cell line from a patient with an ataxia-telangiectasia–like clinical phenotype defective in the p53 response to radiation but with normal ATM activation and efficient downstream phosphorylation of other ATM substrates. No mutations were detected in ATM cDNA. A normal level of interaction between p53 and peptidyl-prolyl-isomerase Pin1 suggests that posttranslational modification was intact in these cells but operating at reduced level. Defective p53 stabilization was accompanied by defective induction of p53 effector genes and failure to induce apoptosis in response to DNA-damaging agents. Continued association between p53 and murine double minute-2 (Mdm2) occurred in irradiated ATL2ABR cells in response to DNA damage, and incubation with Mdm2 antagonists, nutlins, increased the stabilization of p53 and its transcriptional activity but failed to induce apoptosis. These results suggest that ATM-dependent stabilization of p53 and induction of apoptosis by radiation involve an additional factor(s) that is defective in ATL2ABR cells. (Cancer Res 2006; 66(6): 2907-12)
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Introduction
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Ataxia-telangiectasia is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, hypogonadism, and cancer susceptibility (1). Ataxia-telangiectasia cells are defective in their response to double-strand breaks in DNA, manifested by hypersensitivity to ionizing radiation and decreased capacity to activate cell cycle checkpoints after exposure to radiation (2). Ataxia-telangiectasia mutated (ATM), the protein mutated in ataxia-telangiectasia, is present in the nucleus as an inactive dimer and is activated in response to double-strand breaks by autophosphorylation on Ser1981 that causes dissociation of the dimer to form active monomeric forms capable of initiating the phosphorylation of multiple intermediates involved in DNA repair and cell cycle control (3). ATM activation is also controlled by protein phosphatase and acetylase activities (4). ATM is recruited to breaks in DNA by the Mre11 complex (5). Mutations in members of this complex give rise to Nijmegen Breakage Syndrome (Nbs1 mutated) and ataxia-telangiectasia-like disorder (Mre11 mutated). Mutations in both genes decrease the efficiency of activations of ATM compatible with a sensor role for the Mre11 complex (6, 7). The clinical and cellular phenotypes of these disorders overlap with ataxia-telangiectasia (5). We describe here defective DNA damage signaling in an ataxia-telangiectasia-like disorder which is not due to mutations in ATM or the Mre11 complex. Whereas ATM autophosphorylation and signaling to several downstream substrates is normal after DNA damage, these cells are defective in the stabilization of p53 in response to ionizing radiation and fail to undergo apoptosis in response to DNA-damaging and other agents.
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Materials and Methods
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Cell lines and culture conditions. Lymphoblastoid cell lines from control (C3ABR), ataxia-telangiectasia (AT5ABR and AT25ABR), patient (ATL2ABR) as well as control [normal foreskin fibroblasts (NFF)] and patient skin fibroblasts (PSF) were cultured in RPMI 1640 + 10% FCS under standard conditions. Irradiation was done at room temperature using a 137Cs source (Gammacell 40 Exactor, MDS Nordion, 1.1 Gy/min).
Immunoblotting and immunoprecipitation. Immunoblotting and immunoprecipitation were carried out according to supplied protocols (see figure legends). For immunoprecipitations, cells were washed in PBS before resuspension in lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% NP40, protease and phosphatase inhibitors, Benzonase] for 1 hour at 4°C. After centrifugation (16,000 x g, 10 minutes, 4°C), 1 mg of precleared supernatant was incubated (4 hours at 4°C) with 1 µg of antibody and 40 µL of protein G-Sepharose beads (Amersham, Sydney, NSW, Australia). The protein complexes were separated by SDS-PAGE and detected with appropriate antibody.
Protein kinase assay. Specific activity of ATM and Chk2 kinases in vitro was measured as previously described using glutathione S-transferase (GST)-p531-44 and GST-cdc25C as substrates (8).
Immunostaining. Cells were grown on coverslips for 48 hours, irradiated with a single dose of 10 Gy, and fixed in 4% PFA/PBS and permeabilized (0.1% Triton X-100/PBS, 10 minutes, room temperature). Nonspecific binding was blocked before incubating with antibody (in 5% FCS/PBS, overnight, 4°C) before detection with species-specific Alexa Fluor 488 or Alexa Fluor 594 conjugate (Molecular Probes, Eugene, OR). Mre11 foci were counted for at least 160 cells 6 hours after irradiation (8 Gy). Images were captured using a digital camera (Zeiss AxioCamMRm) attached to a fluorescence microscope (Zeiss Axioskop 2plus MOT).
GST pull-down assay. Pin1-GST fusion protein was used as previously described (9). Cells (3 x 107) were lysed in 500 µL lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl and 1 mmol/L EDTA, 1% NP40, 1 mmol/L DTT, protease and phosphatase inhibitors]. Cleared lysate was incubated with GST- or Pin1-GST beads (4 hours at 4°C). After washing beads, proteins were separated by 10% SDS-PAGE and detected using anti-p53 antibody. GST-proteins loaded were visualized by Coomassie staining.
Measurement of apoptosis. Apoptotic bodies were visualized by staining cells with 4',6-diamidino-2-phenylindole (DAPI) after treating with genotoxic agents. For each time point, apoptotic cells were counted from a total of 5,000 cells using a fluorescence microscope (Axioskop 2plus MOT) at x400 magnification. Total cell extracts, 50 to 100 µg, were separated by SDS-PAGE for detection of apoptosis-associated 98-kDa poly(ADP-ribose) polymerase 1 cleavage product. For measurement of DNA damage–induced caspase activity in living cells, a luciferase-based kit (Caspase-Glo 3/7Assay, Promega, Annandale, NSW, Australia) was employed.
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Results
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No defect in ATM activation and signaling in ATL2ABR. Lymphoblasts (ATL2ABR) and skin fibroblasts (PSF) were established from a patient who presented with clinical characteristics of ataxia-telangiectasia. A full clinical picture will be reported elsewhere.6 The amount of ATM protein and radiation-induced ATM kinase activity measured in vitro by phosphorylation of p531-44 was normal in ATL2ABR cells when compared with cells from other family members (Fig. 1A
). Neither ATM protein nor kinase activity was detected in an ataxia-telangiectasia cell line (AT5ABR). ATM is rapidly activated by autophosphorylation on Ser1981 in response to radiation damage (3). The autophosphorylation response in ATL2ABR was comparable to that of a control (C3ABR) cell line in its extent and rate of appearance (Fig. 1B). Whereas ATM protein and kinase activation seemed to be normal in ATL2ABR, it was nevertheless possible that mutation in another protein could compromise radiation-induced ATM downstream signaling. After ATM activation, an early event is the phosphorylation of H2AX (10). This response in ATL2ABR cells was comparable to that in control (Fig. 1C). Ataxia-telangiectasia cells were defective in this response as previously described (10). For efficient activation, ATM is rapidly recruited to the Mre11-Rad50-Nbs1 (MRN) complex at sites of DNA double-strand breaks, where it in turn phosphorylates Nbs1, a member of this complex, to assist in downstream signaling (6, 7). A shift in migration, indicative of Nbs1 phosphorylation after DNA damage, was observed in ATL2ABR (P) cells comparable to that of the other family members (Fig. 1D). In addition, coimmunoprecipitation of Rad50 and Mre11 with Nbs1 confirmed the integrity of the MRN complex in ATL2ABR (P; Fig. 1E) and normal levels of Mre11 foci were induced in these cells after irradiation but there seemed to be fewer foci per cell on average (Fig. 1F). We determined whether another ATM-substrate Chk2 was normally activated in response to radiation in ATL2ABR cells. Normal activation of Chk2 kinase was observed by radiation-induced phosphorylation of a cdc25C substrate determined in vitro and also by mobility shift of Chk2 (Fig. 1G).
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Figure 1. ATL2ABR cells are not defective in radiation-induced ATM signaling. A, ATM kinase activity 1 hour after exposure of cells to 5 Gy of ionizing radiation. Kinase activity was measured in immunoprecipitates of ATM incubated with GST-p531-44 as substrate. ATM loading in the kinase reaction was measured by immunoblotting using a monoclonal anti-ATM antibody. Equal amount of substrate was loaded as shown by Coomassie staining. Ataxia-telangiectasia (A-T) cells that do not express ATM do not show any kinase activity. B, time course of autophosphorylation of ATM on Ser1981 in response to 5 Gy of ionizing radiation. C3ABR, control; ATL2ABR, patient; AT5ABR, ataxia-telangiectasia cell line. Phosphorylation on ATM-Ser1981 was determined using a phosphospecific polyclonal antibody. C, ionizing radiation (IR; 10 Gy, 30 minutes postirradiation) induced H2AX phosphorylation ( H2AX) in control, patient, and ataxia-telangiectasia cells. Actin was used as a loading control. D, ATM-dependent phosphorylation on Nbs1. Phosphorylation of Nbs1 was detected by electrophoretic mobility shift in response to radiation (5 Gy, 1 hour). Actin was used as a loading control. E, integrity of MRN complex in ATL2ABR cells. After immunoprecipitation of the MRN complex using anti-Nbs1 antibody, ATL2ABR cells show comparable amounts of Mre11 and Rad50 to the other family members. F, Mre11 foci formation in control (NFF) and patient (PSF) fibroblasts (8 Gy, 6 hours). The number of foci per cell was determined and plotted in the intervals as indicated. G, activation of Chk2 1 hour postirradiation. Activation was shown in vitro by phosphorylation of a GST-cdc25C substrate. Autophosphorylation in vivo is also shown by a mobility shift of the Chk2 band detected by immunoblotting with anti-Chk2 antibody in response to radiation damage. Cell lines: F, father; M, mother; P, patient; B, sibling; control (NFF) and patient (PSF) fibroblasts. Antibodies used were anti-actin (AC-40, Sigma, Castle Hil, NSW, Australia; 1:1,000), ATM (2CI, GeneTex, San Antonio, TX; 1:1,000), ATM-Ser1981 (Rockland, Gilbertsville, PA; 1:500), Chk2 (07-126, Upstate, Charlottesville, VA; 1:1,000), H2AX (07-164, Upstate; 1:1,000), Nbs1 (NB100-143, Novus; 1:10,000), and Mre11 [12D7, GeneTex; 1:1,000 for Western blot (WB), 1:200 for immunofluorescence].
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Defective radiation-induced p53 response in ATL2ABR. Surprisingly, radiation-induced stabilization of p53 protein was markedly defective in ATL2ABR cells (Fig. 2A
). This was also evident when an antibody detecting phosphorylated p53-Ser15 was employed where a much reduced signal was detectable. Time-course experiments showed that p53 was inefficiently stabilized and, consequently, a much reduced signal for Ser20 phosphorylated p53 was apparent (Fig. 2B). Another posttranslational modification of p53, Lys382 acetylation, was also reduced postirradiation (Fig. 2C). To rule out that this was an artifact of the ATL2ABR cell line, we established fibroblasts (PSF) from the patient for immunofluorescence localization of p53. In response to radiation, p53 nuclear immunoreactivity was greatly enhanced in control fibroblasts (NFF) whereas a weak but discernible response was also present in PSF fibroblasts (Fig. 2D). However, the inefficiently stabilized p53 is correctly localized to the nucleus. Rubbi and Millner (11) have proposed that the nucleolus is a stress sensor responsible for the maintenance of basal levels of p53. Impairment of nucleolar function in response to stress causes p53 stabilization. In response to radiation, immunoreactivity of nucleophosmin, a constituent of the nucleolus, was shown to be reduced in both the patient and control fibroblasts, indicating no defect at this level.
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Figure 2. Defective p53 response in ATL2ABR. A, radiation-induced stabilization of p53 is defective in ATL2ABR cells. p53 stabilization was determined after exposure of cells to radiation (5 Gy, 1 hour) followed by immunoblotting of extracts with anti-p53 antibody. This stabilization is also detected in the extent of Ser15 phosphorylation of p53 using a phosphospecific antibody against this site. B, time course of radiation-induced Ser20 phosphorylation of p53 in C3ABR and ATL2ABR cells. C, time course of radiation-induced Lys382 acetylation of p53 in C3ABR and ATL2ABR cells. Radiation-induced stabilization of p53 protein is also included. D, immunofluorescence detection of p53 and nucleophosmin (NPM) in untreated and irradiated control (NFF) and patient (PSF) fibroblasts 1 hour after irradiation. Cells were fixed with paraformaldehyde, permeabilized, and protein detected with antibodies against p53 and nucleophosmin. Merged images are also presented. E, radiation-induced (5 Gy) Mdm2 and p21 in control (C3ABR) and ATL2ABR cells. F, p53-Mdm2 interaction in response to radiation (5 Gy, 1 hour). Extracts from irradiated and unirradiated cells were immunoprecipitated with an anti-p53 antibody followed by immunoblotting with antibodies against p53 and Mdm2. G, radiation-induced p53 binding to Pin1. For Pin1 pull-down assays, either recombinant GST or GST-Pin1 proteins were incubated with total cell extract from irradiated (after 1 hour) or unirradiated cells. Binding of p53 to Pin1 was confirmed by immunoblotting with an antibody against p53. Cell extracts from control (C1 and C2), ataxia-telangiectasia (ATL2ABR), patient (P), father (F), mother (M), and sibling (B) were used. Antibodies used were anti-actin (AC-40, Sigma; 1:1,000), ATM (2CI, GeneTex; 1:1,000), Mdm2 (SMP14, Serotec, Kidlington, Oxford, United Kingdom; 1:1,000), nucleophosmin (#3542, Cell Signaling, Beverly, MA; 1:500), p53 (pAb1801, Santa Cruz Biotechnology, Santa Cruz, CA, 1:1,000; DO1, BD Biosciences, Brisbane, Qld, Australia, 1:1,000; #9282, Cell Signaling, 1:1,000), p53-Ser15 (#9284, Cell Signaling; 1:500), p53-Ser20 (#9287, Cell Signaling; 1:1,000), and p53-Lys382 (#2525, Cell Signaling; 1:1,000).
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To determine whether the p53 defect observed in ATL2ABR cells was associated with defective downstream signaling, we investigated the induction of a number of p53 effector genes. The results in Fig. 2E show that from 1 to 4 hours postirradiation, p21 and murine double minute-2 (Mdm2) induction is significantly reduced compared with controls. The major mechanism controlling the stability of p53 is its interaction with Mdm2 (12). This interaction leads to the ubiquitination of p53 and its targeting for proteasome degradation (13). Thus, it was possible that this interaction was abnormal in ATL2ABR cells. The constitutive interaction of p53 with Mdm2, as determined by coimmunoprecipitation, was evident in control cells and this association was lost after radiation exposure as expected (Fig. 2F). Constitutive interaction of these two proteins was also observed in ATL2ABR cells but radiation had only a minimal effect on the dissociation. The results suggest that the mechanism involved in releasing p53 from Mdm2 is defective in these cells. The peptidyl-prolyl-isomerase Pin1 has been shown to regulate the function and stability of p53 (9). The interaction between Pin1 and p53 is strictly dependent on the stress-induced phosphorylation and, once formed, causes conformational change in p53 to fulfill its normal biological roles. To determine whether the inefficiently stabilized/phosphorylated form of p53 in ATL2ABR cells had the potential to progress to a functionally active molecule, we evaluated its capacity to interact with Pin1. In response to radiation damage, a normal level of interaction between p53 and Pin1 was observed in ATL2ABR and control cells (Fig. 2E), indicating that all the posttranslational modifications to p53 are intact in these cells but operate at reduced levels.
Resistance to stress-induced apoptosis in ATL2ABR. Because ATL2ABR cells are deficient in p53 stabilization and induction of downstream genes after DNA damage, we determined whether DNA damage–induced apoptosis was deficient. Despite a small but significantly elevated basal level of apoptotic figures (0.1% compared with 0.05% in controls), neither radiation, mitomycin C (MMC), nor H2O2 altered the basal levels of apoptosis in ATL2ABR (Fig. 3A
). On the other hand, all three agents caused a time-dependent increase in apoptosis in control cells over a 48-hour period. The fibroblasts (PSF) from this patient were also resistant to H2O2-induced apoptosis.7 Exposure of cells to genotoxic agents leads to the specific degradation of multiple protein substrates, such as poly(ADP-ribose) polymerase 1, by activation of caspases (14). The results in Fig. 3B show the appearance of the specific 98-kDa poly(ADP-ribose) polymerase 1 cleavage product at both 7 and 16 hours after exposure of control cells to radiation (10 Gy). In ATL2ABR cells, no cleavage product was detected at any time point. Ataxia-telangiectasia cells showed higher basal level of apoptosis as previously reported (15) as poly(ADP-ribose) polymerase 1 cleavage was detected even in untreated cells. Failure to detect cleavage in ATL2ABR cells suggests that caspases are not being activated in these cells in response to DNA damage. The results in Fig. 3C show that there is a significantly greater increase in caspase 3/7 activity in control compared with ATL2ABR in response to a number of genotoxic agents. Because ATL2ABR cells are defective in p53 stabilization and DNA damage–induced apoptosis, we employed nutlins that selectively bind Mdm2 in the p53 binding pocket leading to release and stabilization of p53 (16). The results in Fig. 3D show that the active form of nutlins increased p53 stability in both cell lines after 4 and 8 hours of incubation. A less active enantiomer failed to stabilize p53 as effectively in both cell lines.
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Figure 3. ATL2ABR cells are apoptosis resistant. A, DNA damage–induced apoptosis in control (C3ABR) and ATL2ABR cells. Apoptosis was determined by DAPI staining of apoptotic figures. At least 5,000 cells were counted for each time point up to 48 hours. The DNA-damaging agents used were MMC (0.1 mg/mL), H2O2 (1 mmol/L), and ionizing radiation (10 Gy). B, specific degradation of poly(ADP-ribose) polymerase 1 (PARP1) after exposure of control (C), patient (P), and ataxia-telangiectasia cells to 10 Gy of radiation as a measure of apoptosis. C, comparison of caspase 3/7 activity in response to different genotoxic agents [methylmethanesulfonate (MMS; 2 mmol/L), MMC (0.1 mg/mL), and hydroxyurea (HU; 2 mmol/L)] in control (C3ABR) and ATL2ABR cells at 4 hours after treatment. D, nutlin-induced stabilization of p53 from control (C3ABR) and ATL2ABR cells. Cells were incubated with active (N1) or less active (N0) nutlins for either 4 or 8 hours before preparation of extracts and immunoblotting for p53 and actin as a loading control. E, nutlin induction of Mdm2, p21, and PUMA in control and ATL2ABR cells. Transcriptional activity of p53 was determined by immunoblotting for these p53 effector genes. Active and less active forms of nutlins were employed. F, nutlin-stabilized p53 in C3ABR cells causes apoptosis as determined by poly(ADP-ribose) polymerase 1 cleavage. In ATL2ABR cells, nutlin-stabilized p53 failed to induce apoptosis. G, induction of apoptosis in control and ATL2ABR cells by agents that do not cause DNA damage. Cells were incubated with cycloheximide (CHX; 50 µmol/L) and/or tumor necrosis factor  (TNF; 250 ng/mL) for 20 hours before the apoptosis-associated poly(ADP-ribose) polymerase 1 cleavage product was detected by Western blot. Antibodies used were anti-actin (AC-40, Sigma; 1:1,000), Mdm2 (SMP14, Serotec; 1:1,000), p21 (Ab3, Merck Biosciences, Kilsyth, Vic, Australia; 1:1,000), p53 (pAb1801, 1:1,000), poly(ADP-ribose) polymerase 1 (MCA1522, Serotec; 1:1,000), Puma (Ab1, Merck Biosciences; 1:500), and ß-tubulin (2-28-33, Sigma; 1:1,000).
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It has previously been shown that nutlin-stabilized p53 does not acquire posttranslational modifications that are observed after DNA damage but is nevertheless transcriptionally active (16). We therefore investigated whether incubation with nutlins would lead to transcriptionally active p53 in ATL2ABR cells. Nutlins transcriptionally activated p53, indicated by increased expression of p21, Mdm2, and Puma in both control and ATL2ABR cells (Fig. 3E). Because nutlin-stabilized p53 was transcriptionally active, we determined whether it was also capable of inducing apoptosis. Surprisingly, incubation of ATL2ABR cells with nutlins failed to induce apoptosis as determined by cleavage of poly(ADP-ribose) polymerase 1 (Fig. 3F). At this nutlin concentration, cleavage of poly(ADP-ribose) polymerase 1 was clearly evident in control cells, indicative of apoptosis. These cells seem to be more generally resistant to apoptosis because the non-DNA-damaging agent cycloheximide also failed to induce apoptosis (Fig. 3G).
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Discussion
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On the basis of DNA damage–induced p53 stabilization alone, the defect in ATL2ABR cells distinguishes it from both ataxia-telangiectasia and ataxia-telangiectasia-like disorder. This is supported by mutation analysis of ATM. It seems unlikely that ATL2ABR cells have a mutation in the Mre11 gene because normal activation of ATM was observed in these cells, the Mre11 complex was intact, near normal levels of Mre11 foci were induced by DNA damage, and signaling to downstream substrates such as Chk2 and Nbs1 occurred (7).
Exposure of cells to a variety of genotoxic agents leads to p53 stabilization by a complex series of posttranslational modifications and interactions with other proteins in different subcellular compartments (17). One of the defining characteristics of ATL2ABR cells in this study is the deficiency in stabilizing p53 in response to DNA damage. At short times after radiation, the amount of p53 stabilized is significantly lower than in control cells but it seems to be normally modified (albeit at a lower rate) as judged by the extent of Ser15 and Ser20 phosphorylation and Lys382 acetylation, which are early events in the process of stabilization. Further evidence in support of this emerges from its interaction with Pin1, which is an essential step in the stabilization of the protein, being dependent on p53 phosphorylation on S/P-T sites that induce conformational change, which is required for transcriptional activity (18).
Failure to observe apoptosis in response to DNA damage in ATL2ABR cells is consistent with the defective p53 response. However, even under conditions where p53 was stabilized with nutlins, apoptosis was not induced. This was the case although the p53 effector genes p21, Mdm2, and Puma were induced. These data suggest that the abnormality in ATL2ABR cells is due to a defect in transcriptional-independent p53-mediated apoptosis (19). The actual defect might be due to an imbalance in bcl-2 family members or a defect that reduces the susceptibility of mitochondria to apoptotic stimuli. Whereas the defect in p53 response and apoptosis induction are very evident in ATL2ABR, it remains uncertain about how this relates to the overlapping clinical phenotype of ataxia and oculomotor apraxia with ataxia-telangiectasia. Defective p53 stabilization and p53-Ser15 phosphorylation in response to DNA double-strand breaks is characteristic of ataxia-telangiectasia (2). It seems likely that ATM is responsible for this p53 modification at short times postirradiation. However, in ATL2ABR cells, ATM is normally activated by radiation as evidenced by ATM Ser1981 autophosphorylation and by the ability of ATM to efficiently phosphorylate several downstream substrates with the exception of p53. These data suggest that whereas ATM signaling is normal in these cells, an additional factor is required for p53 stabilization, and they also show that defective signaling in ataxia-telangiectasia cells through these substrates is not responsible for the clinical phenotype that overlaps with that in the patient described here.
We have described here what seems to be a novel form of ataxia overlapping in its clinical and cellular phenotype with ataxia-telangiectasia. The defect is complex in that it relates to both p53 stabilization as well as the capacity of these cells to undergo apoptosis. Further investigation of the defective apoptotic response will assist in understanding the nature of the defect and how it relates to ataxia-telangiectasia. Failure to mount an induced apoptotic response would predict that this patient would be predisposed to genome instability and cancer development.
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Acknowledgments
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Grant support: Associazione Italiana per la Ricerca sul Cancro, Association for International Cancer Research UK, and the Australian National Health and Medical Research Council.
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.
We thank Lyubomir Vassilev (Roche, Brisbane, Qld, Australia) for providing nutlins, Aine Farrell for assistance with cell culture and immunoblotting, and Tracey Laing for manuscript preparation.
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Footnotes
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6 P. Grattan-Smith et al., in preparation. 
7 Unpublished data.
Received 9/23/05.
Revised 1/11/06.
Accepted 1/26/06.
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Abstract
Introduction
