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[Cancer Research 61, 4561-4568, June 1, 2001]
© 2001 American Association for Cancer Research

Regular Articles

Atm Knock-in Mice Harboring an In-frame Deletion Corresponding to the Human ATM 7636del9 Common Mutation Exhibit a Variant Phenotype1

Kevin Spring, Simone Cross, Chung Li, Dianne Watters, Liat Ben-Senior, Paul Waring, Farida Ahangari, Shan-li Lu, Philip Chen, Ihor Misko, Carol Paterson, Graham Kay, Nechama I. Smorodinsky, Yosef Shiloh and Martin F. Lavin2

Queensland Cancer Fund Research Laboratories [K. S., S. C., D. W., F. A., P. C., I. M., C. P., G. K., M. F. L.] and Leukaemia Foundation of Queensland Research Laboratories [C. L., S-l. L.], The Queensland Institute of Medical Research, Herston, Brisbane, Queensland 4029, Australia; Hybridoma Unit, George S. Wise Faculty of Life Sciences [L. B-S.], and Department of Human Genetics, Sackler School of Medicine [N. I. S., Y. S.], Tel Aviv University, Ramat Aviv 69978, Israel; Department of Pathology, University of Melbourne, Parkville, Victoria 3052, Australia [P. W.]; and Department of Surgery, University of Queensland, Herston, Brisbane, Queensland 4029, Australia [M. F. L.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ATM, the gene mutated in the human immunodeficiency disorder ataxia-telangiectasia (A-T), plays a central role in recognizing ionizing radiation damage in DNA and in controlling several cell cycle checkpoints. We describe here a murine model in which a nine-nucleotide in-frame deletion has been introduced into the Atm gene by homologous recombination followed by removal of the selectable marker cassette by Cre-loxP site-specific, recombination-mediated excision. This mouse, Atm-{Delta}SRI, was designed as a model of one of the most common deletion mutations (7636del9) found in A-T patients. The murine Atm deletion results in the loss of three amino acid residues (SRI; 2556–2558) but produces near full-length detectable Atm protein that lacks protein kinase activity. Radiosensitivity was observed in Atm-{Delta}SRI mice, whereas the immunological profile of these mice showed greater heterogeneity of T-cell subsets than observed in Atm-/- mice. The life span of Atm-{Delta}SRI mice was significantly longer than that of Atm-/- mice when maintained under nonspecific pathogen-free conditions. This can be accounted for by a lower incidence of thymic lymphomas in Atm-{Delta}SRI mice up to 40 weeks, after which time the animals died of other causes. The thymic lymphomas in Atm-{Delta}SRI mice were characterized by extensive apoptosis, which appears to be attributable to an increased number of cells expressing Fas ligand. A variety of other tumors including B-cell lymphomas, sarcomas, and carcinomas not seen in Atm-/- mice were observed in older Atm-{Delta}SRI animals. Thus, expression of mutant protein in Atm-{Delta}SRI knock-in mice gives rise to a discernibly different phenotype to Atm-/- mice, which may account for the heterogeneity seen in A-T patients with different mutations.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human genetic disorder A-T3 is characterized by immunodeficiency, neurodegeneration, sensitivity to ionizing radiation, and cancer predisposition (1 , 2) . Chromosomal instability in this disease is characterized by abnormal rearrangements involving chromosomes 7 and 14 in the vicinity of the TCR and immunoglobulin genes (3 , 4) . It has been suggested that a reduced capability in processing double-strand breaks in DNA is responsible for the radiosensitivity, immunodeficiency, infertility, and cancer predisposition (5 , 6) . Evidence in support of this has been provided by disruption of the Atm gene in mice. These animals developed malignant thymic lymphomas by 4–5 months of age (7, 8, 9) , and these lymphomas had translocations in chromosome 14 occurring in both alleles of the TCR {alpha}/ß locus (10) . These data suggest that breaks occurring within the TCR locus during T-cell development undergo inappropriate end-joining, giving rise to genome instability and cancer predisposition. Consistent with this, Liao and Van Dyke (11) failed to observe the development of thymic lymphomas in Atm-/- Rag1-/- mice in which V(D)J recombination had been disrupted. On the other hand, Petiniot et al. (10) reported a lower frequency and longer latency period for thymic lymphomas in Atm-/- Rag2-/- mice compared with Atm-/- mice. Overall, the results point to an important but nonessential role for V(D)J recombination in tumorigenesis in Atm-deficient thymocytes (10) .

The phenotype observed in Atm-/- mice generally reflects that seen in A-T patients with the exception of neurodegeneration, where the majority of studies failed to reveal such abnormalities (7, 8, 9 , 12) . In two cases, some evidence for neuronal degeneration and abnormal development of Purkinje cells was provided in Atm-/- mice (13 , 14) . In all of the Atm-/- mice generated to date, the disruption of the gene was achieved by means of gene inactivation leading to complete loss of Atm protein expression (7, 8, 9 , 12 , 14) . Mice on either inbred or mixed genetic backgrounds did not show any phenotypic variation (7) . Disruption of Atm led to a smaller size at birth, body weights of these mice were reduced during the early growth period, and fibroblasts from these animals grew poorly (7, 8, 9) . Although the architecture of various lymphoid tissues was normal in Atm-/- mice, these organs were generally smaller in size (7, 8, 9) .

Although there are clear hallmarks that characterize the A-T phenotype, some variability exists in these features (1) , which might be explained by the nature of the mutation or genetic background. In addition to a phosphatidylinositol 3-kinase domain, the ATM protein contains several regions that interact with other proteins or are predicted to be functionally important (15 , 16) . Furthermore, there is evidence for a dominant-negative effect of a region of the ATM protein containing a putative leucine zipper motif (17) . Because up to 20% of A-T patients express mutant protein and none of the Atm-/- mice produced to date express Atm protein, it was important to generate a mouse expressing mutant Atm protein and to determine whether these mice display any variation in the characteristics associated with the A-T phenotype. Accordingly, we produced mice homozygous for a nine-nucleotide in-frame deletion (7666del9) in Atm, which was predicted to give rise to a protein with three amino acids deleted (SRI; 2556–2558). This mutant corresponds to one of the most common A-T mutations found to date (15 , 18) . We report here that the phenotype of Atm-{Delta}SRI mice is significantly different to that described for Atm-/- mice.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Gene Targeting and Generation of Atm-{Delta}SRI Mice.
To generate a mutant mouse model of the human 7636del9 mutation found in A-T patient AT1ABR (15 , 18) , we used homologous recombination and the Cre-loxP system to introduce a nine-nucleotide in-frame deletion into exon 54 of mouse Atm (19) . The gene targeting vector was constructed using a 5.5-kb EcoRV-PstI genomic fragment isolated from a {lambda}ZAP2 clone containing a mouse Atm insert derived from 129/SvJ genomic DNA ({lambda}DA1.1). On the basis of the mouse Atm cDNA sequence, removal of nucleotides 7666–7674 resulted in deletion of three amino acid residues (SRI; 2556–2558) at a position corresponding to the human 7636del9 mutation from patient AT1ABR. Site- directed mutagenesis was used to introduce this mutation into exon 54.

A 36-bp mutagenesis primer (5'-CTGATAGCTAATCTCACTTGATCACCCCCATCATAC-3') was used to delete nucleotide 7666–7674 and also to create a novel BclI restriction site neutral polymorphism at nucleotides 7680. This restriction site was included as a marker to track the deletion mutation. A second 29-bp mutagenesis primer (5'-GTAACTGCAAGGGCCCTTTTGTGTTGTG-3') was also used to introduce a unique ApaI site within intron 54, into which a loxP-neo/gpt-loxP selection cassette was inserted (Fig. 1)Citation . After transfection into 129SvJ ES cells (C1368) and G418 selection, two independently targeted clones were injected into C57BL/6J blastocysts, and 10 high-grade chimeric males were produced. These were, in turn, backcrossed with C57BL/6J, and of these, 8 transmitted the mutation through the germ line. The floxed neo/gpt selection marker was removed subsequently by crossing heterozygotes with the EIIa-Cre deleter mouse strain (20) . The Cre-deleted mutant Atm mice were maintained on a 129/SvJ:C57BL/6J background. Mice generated by intercrossing heterozygotes were genotyped by Southern blot and PCR analysis at postnatal day 10. Tail tip DNA was extracted using proteinase K digestion and NaCl precipitation. Southern blotting was carried out by standard procedures. PCR genotyping was performed using forward (5'-TCTCATGTATCAATTGGCTGCTGC-3') and reverse (5'-AATTGTTAACCAATTCTGGGTGGC-3') primers to amplify exon 54 (wild-type 663-bp and mutant 714-bp fragments). Cycling conditions were 94°C for 12 min, 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min over 35 cycles and analyzed on 1.2% agarose gels.



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  		Fig. 1. Strategy used to mutate the mouse Atm via gene targeting. a, wild-type Atm with exons 52–56 marked and the targeting vector after mutating nucleotides 7666–7674 to introduce a nine-nucleotide deletion within exon 54. The position of the floxed neo/gpt cassette in intron 54 is also shown. Mutated Atm was detected in ES cells after digestion with EcoRV and DNA blot analysis using probe A. Deletion of the neo/gpt cassette in offspring from mating Atm-{Delta}SRI heterozygous mice with EIIa-Cre deleter mice was detected by PstI digestion and hybridization with probe B. b, Southern blot analysis of tail tip DNA prepared from wild-type (+/+), Atm-{Delta}SRI heterozygous (+/{Delta}SRI), and Atm-{Delta}SRI homozygous ({Delta}SRI/{Delta}SRI) mutant mice. DNA was digested with EcoRV and probed with probe A. The expected size of the EcoRV fragments detected by probe A are 9.2 and 5.9 kb for the wild-type and mutant alleles, respectively. c, PCR and restriction-digestion analysis of tail DNA from +/+, +/{Delta}SRI, and {Delta}SRI/{Delta}SRI Atm-{Delta}SRI mice. Primers flanking exon 54 amplify either a wild-type 663-bp fragment or a mutated 714-bp fragment encompassing the nine-nucleotide deletion, BclI restriction site, and single loxP sequence residing in intron 54 (top panel). BclI restriction digestion of the genomic PCR products results in cleavage of the mutated allele fragment to two bands of 354 and 360 bp (bottom panel). d, sequence analysis of PCR fragments derived from wild-type and homozygous Atm-{Delta}SRI mice. Shading, the nine nucleotides deleted in the mutant Atm allele; *, nucleotide substitution used to create the BclI site.

 
Mouse Growth Determinations and Survival.
Mice were weighed at 7-day intervals beginning at postnatal day 10 to day 45 of age. After weaning, mice were divided by gender, and both food and water were provided ad libitum. Analysis of survival time for wild-type Atm-/- (kindly provided by Philip Leder, Harvard University, Boston, MA) and Atm-{Delta}SRI was calculated from date of birth to date of death or date last known alive. In this study, 129SvEv inbred (Atm-/-) and 129SvJ:C57BL/65 mixed background (Atm-/- and Atm-{Delta}SRI) mice were used. Mice exhibiting ill health or obvious tumor burden were killed, and the underlying morbidity was recorded as the cause of death. Kaplan-Meier survival curves were generated for death attributable to tumors of either thymic or other tumor types, using the SPSS statistical package. Statistical significance was measured using the log-rank test at the 0.05 level.

Flow Cytometry Analysis.
Thymocytes and lymph node cells from wild-type Atm-/- and Atm-{Delta}SRI mice were immunophenotyped by surface immunofluorescent labeling and flow cytometry. Cells were disaggregated in RPMI 1640 containing 10% FCS, 2 mM glutamine, and 5.5 x 10-5 M 2-mercaptoethanol. Cells (1.0–1.5 x 105) in 20 µl of PBS with 1% FCS were incubated at 4°C for 30 min with 2–4 µl of antimouse mAbs, washed once in PBS + 1% FCS, and then analyzed for three-color immunofluorescence (FITC, phycoerythrin, and TRIC) with a Becton Dickinson FACScan and CellQuest software (version 3.1f). Cells expressing surface CD3, CD4, CD8, CD62L, CD44, {alpha}/ßTCR (Caltag Laboratories Inc., Burlingame, CA), Fas/CD95, FasL (PharMingen, San Diego, CA), and Annexin V-FITC (PharMingen) are presented as a percentage of the total number of cells. Thymic lymphomas were disaggregated and analyzed in the same way.

Production of mAbs.
The mAb MAT3-4G10/8 was raised against a peptide spanning positions 1967–1988 of murine Atm, to which a cysteine residue was added at the NH2 terminus for coupling to keyhole limpet hemocyanin. Mice were immunized with 50 µg of antigen (total of six injections over 15 weeks), and the appearance of antigen-specific antibodies was monitored by ELISA based on the antigen peptide and then by immunoblotting of mouse tissue extracts and immunoprecipitation of Atm from these tissues. Hybridomas were established using standard methods, and antibody production was monitored as described above. Seventeen clones were finally stocked, and clone 4G10/8, which produces heavy chain IgG1, was used in this study. The antibody was purified using fast protein liquid chromatography over a HiTrap protein G column (Pharmacia Biotech) and dialyzed against PBS.

Western Blotting.
Protein extracts were prepared from spleens by homogenization in TGN buffer [50 mM Tris (pH 7.5), 50 mM ß-glycerophosphate, 150 mM NaCl, 10% glycerol, 1% Tween 20, 1 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin, 5 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM DTT] and centrifuged at 13,000 x g for 15 min. Protein concentrations were determined using the Bio-Rad DC protein assay kit according to the manufacturer’s recommendations. Protein samples (100 µg) were separated on 5 or 10% denaturing gels and blotted onto Hybond-C (Amersham) nitrocellulose membranes. After blocking in 4% milk powder, 0.1% Tween 20, and PBS for at least 1 h, blots were incubated for 1 h at room temperature or overnight at 4°C with the relevant primary antibody. Immunoreactive bands were visualized with Renaissance chemiluminescence substrate and captured on Kodak XAR X-ray film. The primary antibodies used in this study were mouse mAb anti-Atm MAT3-4G10/8 and rabbit antiactin (Sigma Chemical Co.).

In Vitro Atm-Kinase Assay.
Atm-kinase activity was determined using the method described by Canman et al. (21) . For whole cell lysate isolation, cells were lysed on ice in TGN buffer. After centrifugation at 13,000 x g for 15 min, 1 mg of extract was precleared with protein A-Sepharose beads. Atm was immunoprecipitated with Atm antibody (MAT3), and the kinase activity was determined.

Cell Survival.
Thymi were dissected from mice, and thymocytes were disaggregated in RPMI 1640 (106/ml) as described above. Cells were incubated with or without IL-2 (60 µg/ml) at 37°C in an atmosphere of 5% CO2 for 24 h. Cells were exposed to ionizing radiation (0–4 Gy), and cell viability was determined by adding 0.1 ml of 0.4% trypan blue to a 0.5-ml suspension (22) . The number of viable cells was determined at 48 and 72 h after irradiation.

Induced Chromosome Aberrations.
Cells were irradiated with 1 Gy of {gamma}-rays. For G2-phase cells, Colcemid (final concentration, 0.1 mg/ml) was added immediately after irradiation, 1–2 h before harvesting. The cells were treated for 15 min in 0.075 M KCl, fixed in methanol:glacial acetic acid, 3:1 (vol/vol), and spread on glass slides. The cells were then stained with Giemsa, and 50 metaphases were analyzed for each sample (22) .

ISEL of Tumor Sections.
Paraffin sections of thymoma from Atm-/- and Atm-{Delta}SRI mice were dewaxed, digested with pepsin, and subjected to ISEL, essentially as described by Ansari et al. (23) using Biotin-16-dUTP (Enzo Diagnostics) in the labeling mix. Incorporated biotin-dUTP was detected with horseradish peroxidase coupled to streptavidin (DAKO) and 3,3'-diaminobenzidine staining. The sections were counterstained with hematoxylin, mounted, and photographed using an Olympus BH2 microscope.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of Atm-{Delta}SRI Mutant Mice Expressing Mutant Atm Protein.
Because the Atm protein is a high molecular weight molecule containing several potential domains in addition to the kinase domain, it is important to establish whether the presence of mutant Atm protein influences the A-T phenotype. To date, all of the Atm mouse models produced result in Atm gene inactivation and a complete abrogation of Atm protein expression. We therefore generated mice homozygous for a nine-nucleotide in-frame deletion (7666del9) in Atm resulting in the loss of three amino acids (SRI; 2556–2558), hereafter referred to as Atm-{Delta}SRI. The strategy used to introduce the {Delta}SRI mutation into Atm included the insertion of a loxP-flanked selectable marker cassette into the intron downstream of exon 54 (Fig. 1aCitation ; Ref. 24 ), which contains the nine-nucleotide deletion. Chimeric mice generated from two independently targeted 129SvJ ES cell clones (D11A and C9B) were crossed with C57BL/6J mice to produce Atm-{Delta}SRI heterozygotes. The loxP-selectable marker cassette was removed by crossing Atm-{Delta}SRI heterozygous mice with an EIIa-Cre deleter mouse strain that constitutively expresses Cre recombinase (20) . Atm-mutant mice were identified by Southern blot analysis of tail tip DNA (Fig. 1b)Citation or PCR analysis of a region containing the deletion mutation and BclI site (Fig. 1cCitation , top panel). Using this analysis, it is possible to distinguish Atm-{Delta}SRI homozygotes (714 bp) from heterozygotes and wild type (663 bp). The increased size of the Atm-{Delta}SRI homozygous band is attributable to the presence of a single loxP site in intron 54. Cleavage with BclI confirmed the presence of the mutation (Fig. 1cCitation , bottom panel). Deletion of the nine-nucleotide sequence was verified by genomic DNA sequencing (Fig. 1d)Citation . These {Delta}SRI mutant mice expressed near full-length Atm protein, whereas no Atm protein was detected in Atm-/- mice, as determined by immunoblotting of spleen cell extracts with a mAb (MAT 3) derived against the mouse Atm sequence (Fig. 2a)Citation . Because we have shown previously that the corresponding human mutant protein typified in the cell line AT1ABR (15 , 18) is kinase-dead and thereby incapable of phosphorylation of p53 on serine 15 (25) , we measured the kinase activity of Atm-{Delta}SRI splenocytes using p531–40 as a substrate as described previously (21 , 26) . A marked increase in Atm-kinase activity was observed in wild-type spleen cell extracts in response to radiation exposure, whereas only a low level of residual kinase activity, which did not respond to radiation, was observed in Atm-{Delta}SRI extracts (Fig. 2b)Citation . As expected, extracts from Atm-/- mice displayed no Atm-kinase activity.



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  		Fig. 2. Expression and activity of Atm protein in splenocytes from wild-type, Atm-/-, and Atm-{Delta}SRI mice. a, immunoblotting of Atm protein (100 µg) was carried out using a mAb (MAT3) raised against a peptide corresponding to amino acids (1967–1988) of murine Atm (see "Materials and Methods"). Samples were separated on 4.5% SDS-PAGE prior to blotting, and protein loading was determined with an antiactin antibody. b, Atm kinase activity as determined by immunoprecipitation with anti-Atm (MAT3) antibodies. GST-p531–40 was used as a substrate. Extracts were prepared from unirradiated and irradiated (IR) cells (10 Gy) 1 h after exposure, and 1 mg immunoprecipitated with antibody prior to the kinase reaction. The amount of GST-p531–40 in each sample was determined by Coomassie staining. WT, wild type.

 
Growth and Other Defects in Atm-{Delta}SRI Mice.
Previous data have revealed the presence of a growth defect in Atm-/- mice, which appears to be largely attributable to disruption of the gene per se (7, 8, 9) . Consistent with these observations, growth retardation was seen both in male (20% reduction) and female (18% reduction) Atm-{Delta}SRI mice from birth to 45 days of age and persisting throughout adulthood. A-T cells and ES cells from Atm-/- mice have been shown to be hypersensitive to ionizing radiation (14 , 27 , 28) . Incubation of freshly isolated thymocytes from Atm-{Delta}SRI and Atm-/- mice in the presence of IL-2 for 24 h before radiation exposure (1–4 Gy) resulted in significantly more cell death than observed with wild-type thymocytes at 48 h after irradiation (Fig. 3Citation , top panel). There appeared to be somewhat less killing in the absence of IL-2, but thymocytes were still distinguishable from wild-type (results not shown). The extent of cell death was similar in Atm-{Delta}SRI and Atm-/- thymocytes. This was additionally confirmed by preparing metaphase chromosomes from irradiated thymocytes. The number of induced chromosome aberrations/metaphase was approximately one in wild-type thymocytes, whereas the number for Atm-{Delta}SRI and Atm-/- mice was approximately three times that value (Fig. 3Citation , bottom panel). We also demonstrated enhanced radiosensitivity in splenocytes from Atm-{Delta}SRI and Atm-/- mice (data not shown). These data are comparable with the relative values observed in irradiated A-T and normal cells (22) .



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  		Fig. 3. Survival of thymocytes from wild-type, Atm-/-, and Atm-{Delta}SRI ({Delta}SRI) mice in response to radiation. Thymocytes were isolated and incubated in medium supplemented with IL-2 (60 µg/ml) for 24 h before radiation exposure. Top panel, viability was determined by trypan blue exclusion at 48 and 72 h after irradiation. The viability data at 3 days after irradiation are depicted. Bottom panel, radiation-induced chromosome aberrations in wild-type, Atm-/-, and Atm-{Delta}SRI thymocytes isolated and treated as described above. The radiation dose used was 1 Gy.

 
Although neurological abnormalities have been reported for Atm-/- mice, the majority of the evidence suggests that neurodegeneration is not present in these animals (7, 8, 9 , 12) , with the exception of two reports where degeneration of several different types of neurons in the cerebellar cortex of Atm-/- mice was evident from electron microscopic analysis, in one case (13) , and in the other, where ectopic expression and abnormally differentiated Purkinje cells were apparent (14) . As with Atm-/- mice, Atm-{Delta}SRI mice, 2–16 months of age, failed to show any evidence of gross neuronal degeneration, specific loss, or abnormal migration of Purkinje cells (results not shown). Similar to Atm-/- mice, there was also evidence of testicular abnormalities (poorly developed testes) and disruption of spermatogenesis in Atm-{Delta}SRI mice (n = 2). In contrast to seminiferous tubules of testes from wild-type mice where abnormalities were not observed, tubules from Atm-{Delta}SRI mice were disrupted with evidence of degeneration of spermatocytes and loss of spermatids (Fig. 4Citation , a and b). Similarly, oogenesis in Atm-{Delta}SRI mice (n = 2) was disrupted with an observed lack of maturing follicles and oocytes (results not shown).



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  		Fig. 4. Effect of Atm disruption on reproductive capability. H&E-stained sections through seminiferous tubules from wild-type (a) and Atm-{Delta}SRI mice (b). x40.

 
Immunological Abnormalities in Atm-{Delta}SRI Mice.
A variable but common characteristic in A-T is immunodeficiency and immune defects, which are reproduced in Atm-/- mice. To characterize the T-cell development/phenotype in Atm-{Delta}SRI mice, cell samples of thymus and inguinal lymph node were analyzed by flow cytometry and compared with those from age-matched (7–12 weeks) wild-type and Atm-/- mice. Atm-{Delta}SRI mice had smaller thymi, spleens, and lymph nodes than wild-type mice and 10 times fewer thymocytes than wild-type mice, in agreement with data for Atm-/- mice. A reduction of 30–50% in the total number of thymocytes expressing {alpha}/ßTCR and CD3 was observed in the thymi of Atm-/- and Atm-{Delta}SRI mice, which is consistent with previous results for Atm-/- mice (9) . More detailed analysis of eight 12-week-old Atm-{Delta}SRI mice revealed that three of these mice showed significant increases in cell size, as reflected in scatter analysis and apparently random accumulation of either CD4-/Low CD8-/Low (60%), CD8+ (39%), or CD4+ (64%) SP populations. This heterogeneity in three Atm-{Delta}SRI mice is illustrated in Fig. 5aCitation . These changes in T-cell subtypes were also evident in two younger (8-week) Atm-{Delta}SRI mice. On the other hand, Atm-/- mice more closely resembled the wild type in the proportions of DP and SP T cells.



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  		Fig. 5. FACS analysis of T-cell surface antigen expression in normal thymocytes and thymic lymphomas. a, forward scatter analysis (top panel) of thymocytes from wild-type, Atm-/-, and Atm-{Delta}SRI mice. Bottom panel, surface expression for CD4 and CD8 T-cell populations. Top right quadrants, double-positive cell populations. b, surface expression of CD4 and CD8 on thymic lymphomas from Atm-/- and Atm-{Delta}SRI mice.

 
The lymph nodes of Atm-/- mice contain only 50% of lymphocytes expressing either CD3, CD4+CD8+, and CD62L or CD44 compared with Atm-{Delta}SRI and wild-type mice (results not shown). On the basis of the combination of low CD3, low CD62L, and low CD44 expression, the Atm-/- lymph nodes appear to consist primarily of T cells at an early stage of development (29) . Both CD62L and CD44 are putative markers for memory T cells in mice.

Life Span, Thymic Lymphomas, Apoptosis, and Up-Regulation of FasL in Atm-{Delta}SRI Mice.
Patients with A-T have been demonstrated to develop a spectrum of tumors, primarily leukemias and lymphomas (6) . On the other hand, Atm-/- mice develop exclusively malignant thymic lymphomas and die between 2 and 5 months of age when held in non-SPF facilities (7, 8, 9 , 28) . In this study (under non-SPF conditions), the life span of Atm-{Delta}SRI mice (n = 43) was increased dramatically compared with Atm-/- mice (n = 26). When 100% of Atm-/- mice were dead, only 50% of Atm-{Delta}SRI animals had died (Fig. 6)Citation . At this stage, autopsies revealed the presence of thymic lymphomas in all of these animals, indicating that death was a consequence of malignancy. Almost 30% of Atm-{Delta}SRI mice were alive after 16 months. The results obtained here for Atm-/- mice are consistent with previous reports on survival of these animals (9 , 30) . Difference in survival appears not to be attributable to genetic background because both types of mice used here were of mixed genetic background: (a) C57BL/6J crossed with 129SvJ for Atm-{Delta}SRI; and (b) C57BL/6J crossed with 129SvEv for Atm-/-. Differences between 129Sv substrains do not significantly affect tumor incidence (31) , and previous results have failed to demonstrate phenotype variance between mixed and inbred backgrounds for Atm-/- mice (7) .



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  		Fig. 6. Kaplan-Meier survival plot of wild-type (WT), Atm -/-, and {Delta}SRI mice. P was calculated by comparing life spans of mice from each genotype using the SPSS statistical package. Atm-/- and Atm-{Delta}SRI mice of either 129Sv inbred or 129Sv:C57BL/6J mixed genetic background were used in this study. No differences in survival were observed with mice of different genetic backgrounds.

 
Phenotypic analysis of the thymic lymphomas revealed that the Atm-/- phenotype was characterized predominately by CD4+CD8+ DP cells and low CD3 (data not shown), whereas the Atm-{Delta}SRI lymphomas were more diverse, reflecting the greater heterogeneity of DP and SP populations of T cells seen in the thymi of these animals and may represent polyclonal tumors (Fig. 5b)Citation .

Morphological examination of thymic lymphomas from Atm-{Delta}SRI mice (>3 months of age) revealed that these tumors were more spongy and the cells less tightly aggregated than in the Atm-/- tumors. Histological examination of tumors showed that there was a significantly increased proportion of cells undergoing what appeared to be spontaneous apoptosis based on nuclear fragmentation in Atm-{Delta}SRI tumors (Fig. 7b)Citation compared with Atm-/- tumors (Fig. 7a)Citation . These cells were confirmed as apoptotic by ISEL staining (Fig. 7Citation , c and d), and ~50% of Atm-{Delta}SRI thymic lymphoma cells were Annexin V positive compared with ~15% in Atm-/- thymic lymphomas (results not shown). To investigate these differences, we isolated disaggregated cells from the two types of tumors as well as thymocytes from wild-type and from the Atm-disrupted mice and performed flow cytometric analysis to compare surface markers for susceptibility to undergo apoptosis (32) . Down-regulation of the Fas receptor (Fas/CD95) was evident in thymocytes in both Atm-/- and Atm-{Delta}SRI mice, with 30–40% fewer Fas-positive cells than in wild type (Table 1)Citation . This pattern of down-regulation is also seen in thymic lymphomas from both forms of mutant mice. Expression of FasL was approximately the same in thymocytes from wild-type and mutant mice (Table 1)Citation . A small increase was evident in thymic lymphomas from Atm-/- mice, but in thymic lymphomas from Atm-{Delta}SRI mice there was an ~14-fold increase in the number of cells expressing FasL (Table 1)Citation . This corresponds well with the increased extent of apoptosis detected in thymic lymphomas from Atm-{Delta}SRI mice compared with Atm-/- mice. However, this extent of apoptosis was not observed, and FasL was not up-regulated in Atm-{Delta}SRI mice developing lymphomas at <2 months of age. To test the functionality of the FasL expression in Atm-{Delta}SRI tumors, we incubated thymic lymphoma cells with a target cell line AT1ABR, a lymphoblastoid cell line from a patient with A-T, containing the same mutation as in the Atm-{Delta}SRI mice (18) . This cell line has a high expression of Fas.4 At a ratio of 3:1 lymphoma:target cells, viability of AT1ABR was reduced by 80% after 12-h incubation demonstrating that the up-regulated FasL was active in the apoptotic process observed in Atm-{Delta}SRI tumors (results not shown). This was substantiated additionally by attempts to establish the tumor lines in culture, where it was observed that those from Atm-{Delta}SRI were more difficult to grow.



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  		Fig. 7. Histological examination of thymic lymphomas. a and b, H&E staining of thymic lymphoma sections from Atm-/- and Atm-{Delta}SRI mice. x40. The Atm-/- thymic lymphoma (a) shows a reduced nuclear fragmentation indicative of apoptosis as compared with the Atm-{Delta}SRI thymic lymphoma (b), which shows extensive apoptosis. c and d, ISEL of paraffin sections of thymic lymphomas from Atm-/- and Atm-{Delta}SRI mice. Cells were labeled with Biotin-16-dUTP, and apoptosis was detected with horseradish peroxidase-coupled streptavidin and 3,3'-diaminobenzidine.

 

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  		Table 1 Flow cytometric analysis of Fas and FasL in Atm-/- and Atm-{Delta}SRI thymic lymphoma

 
Development of a Spectrum of Tumors in Long-lived Atm-{Delta}SRI Mice.
It is evident from the data in Fig. 6Citation that the longevity of Atm-{Delta}SRI mice is markedly greater than that of most Atm-/- mice. As indicated above, all of the Atm-/- mice had died from thymic lymphomas by 40 weeks in this study. The 44% (19 of 43) of Atm-{Delta}SRI mice that had died by the same time also died with the same tumors. Very few Atm-{Delta}SRI mice died after this time from thymic lymphomas, but a significant number of deaths (7) were associated with a variety of other tumors. Upon autopsy, the mice usually displayed marked splenomegaly but with minimal involvement of the thymus. Immunotyping showed that three of these animals had B-cell leukemia. The B-cell tumors grew aggressively with no evidence of FasL up-regulation. Furthermore, histopathological examination of various tissues/organs revealed the presence of multiple tumor types including ovarian granulosa cell tumors, epithelial carcinomas, histiocytic/reticulum cell tumors, ovarian sex cord, and stromal cell tumors. Mutant Atm protein was expressed in both lymphoid and solid tumors (results not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The Atm-{Delta}SRI mouse represents a dysfunctional Atm model that expresses mutant protein unlike all of the other Atm-/- mice described to date, which do not express Atm protein because of the nature of the gene disruption (7, 8, 9 , 12 , 14) . In one case, a neor gene replaced a portion of the Rad-3 homology domain of Atm, but neither full-length Atm transcript nor protein were detected (14) . The mutant mouse described here corresponds to a commonly observed mutation in A-T patients (7636del9; Refs. 15 and 18 ). As with the human mutant, Atm-{Delta}SRI protein is less stable as judged by the amount of Atm determined by immunoblotting, and no Atm-kinase activity was detected, similar to that for the human mutant (33) . However, some variability in the amount of Atm-{Delta}SRI protein was observed in different experiments. Because this short deletion is upstream from the kinase domain, it is likely that it causes an inactivating, conformational change in the Atm protein or prevents binding of a protein critical for activity. Since the SRI sequence is in a putative domain, FAT (Ref. 34 ; FRAP, ATM, and TRAPP), present in a subfamily of phosphatidylinositol 3-kinases, and because this domain is implicated in multimeric protein complex formation, its loss may interfere with protein-protein interactions. There is evidence that ATM is present in a large protein complex with BRCA1, BLM, Mrell-Rad50-NBS1, and DNA repair proteins, called BRCA1-associated genome surveillance complex (35) , but none of these proteins have been shown to interact with this region of ATM.

In this study, thymocytes and splenocytes from both Atm-/- and Atm-{Delta}SRI mice were more susceptible to radiation-induced killing than wild-type cells. This was confirmed by radiation-induced chromosome aberrations, which were 3-fold higher in both types of mutant mouse thymocytes than in wild-type cells. This is in agreement with two previous reports that showed that ES cells from Atm-/- mice were hypersensitive to radiation at low doses (>=5 Gy; Refs. 14 and 28 ), although other reports suggest that Atm-/- cells are only equally as sensitive or even less sensitive than controls to ionizing radiation (12 , 28 , 30) . However, at least in the case of embryonic fibroblasts, extremely poor growth makes it difficult to differentiate between the two cell types (7) . Although Elson et al. (9) have shown that the relative extent of the increase in chromosome damage by bleomycin is similar in Atm-deficient and wild-type murine fibroblasts, the absolute value, 9.71 breaks/metaphase, is considerably greater in Atm-/- cells than in wild type (1.70 breaks/metaphase). This might very well translate into increased cell killing. Indeed, Westphal et al. (36) have shown that loss of Atm radiosensitizes multiple cell types including fibroblasts, bone marrow, and gastrointestinal cells as well as p53-null bone marrow cells. Furthermore, whereas p53-/- cells are considered generally to be radioresistant, mitogenic stimulation of p53-/- lymphocytes renders them susceptible to radiation-induced killing (37) . In other studies, exposure conditions were different in that whole animal irradiation occurred prior to cell isolation, higher doses were used (5–20 Gy), and apoptosis was the end point (12 , 28 , 30 , 36 , 38) . All of these studies point to reduced apoptosis in irradiated Atm-/- cells. However, because death by apoptosis may only account for 30% of radiation-induced killing in A-T cells (39) , the data obtained may not reflect the actual extent of cell killing. In addition, Brown and Wouters (40) have reported that when clonogenic survival is used as an end point of cell killing, ability to undergo apoptosis does not contribute significantly to the sensitivity of tumor cells to radiation and chemotherapeutic drugs.

Although a number of the Atm-/- phenotypic characteristics have been reproduced in the Atm-{Delta}SRI mice, the most dramatic difference is the significantly greater life span of these animals. In the experiments conducted here, Atm-{Delta}SRI and Atm-/- mice were bred in a non-SPF facility. In previous reports, Atm-/- mice were also housed under non-SPF conditions, and all of the animals had died with thymic lymphomas between 2 and 5 months of age (7, 8, 9 , 30) . The Atm-/- mice used here died at rates comparable with those observed previously for these animals (9) . Longer life spans in Atm-/- mice appear to be attributable to breeding in pathogen-free conditions. This is particularly evident with the Atm-/- mice produced by Barlow et al. (7) , where the mice did not survive beyond 4.5 months, but when the same animals were maintained in SPF housing, 40% of animals were still alive after 18 months (10) . Breeding was also carried out in pathogen-free facilities by Borghesani et al. (14) , where 50% of Atm mutant mice were alive after 10 months. Clearly, SPF conditions were not required for the prolonged life span described here in Atm-{Delta}SRI mice.

The extent of apoptosis occurring in thymic lymphomas from Atm-{Delta}SRI mice may contribute to the longevity in Atm-{Delta}SRI mice. Previous studies have failed to observe apoptotic cells in tumors from the Atm-/- mice used in the present investigation (41) . However, thymic lymphomas derived from Atm/p21 double-null mice show high levels of apoptotic cells. It appears likely that loss of p21 potentiates the apoptotic response presumably because of a lack of G2 arrest (41) . It seems likely that the increased apoptosis seen here in Atm-{Delta}SRI tumors is attributable to up-regulation of FasL. Interaction of FasL with the death receptor Fas is responsible for the subsequent assembly of a death-inducing signaling complex, which in turn activates caspase-8 and other downstream caspases leading to cell death (42 , 43) . FasL is up-regulated to prevent uncontrolled T-cell expansion and cytokine production (44) but can also be up-regulated by a variety of other agents including ionizing radiation (45) , reactive oxygen species, IFNs, tumor necrosis factor-{alpha} (46 , 47) , and nuclear factor-{kappa}B (48) . An imbalance in any of these factors caused by the presence of mutant Atm could contribute to the FasL up-regulation. The increased expression of FasL in Atm-{Delta}SRI thymic lymphomas and consequent apoptosis may explain why only 50% of Atm-{Delta}SRI mice succumb to thymic lymphomas after 40 weeks, at which time all of the Atm-/- mice (n = 26) in this study had died from thymic lymphomas (Fig. 6)Citation . The "take rate" of these tumors may be reduced by Fas/FasL-induced apoptosis. In all, 19 of 43 (44%) of Atm-{Delta}SRI mice died of thymic lymphomas up to 10 months of age. Some of the other animals died from splenetic, ovarian, and B-cell tumors with evidence of metastatic spread. To date, no B-cell tumors have been recorded in Atm-/- mice, and there has been no evidence of tumor metastases in these mice. Whereas the Atm-{Delta}SRI mice have significantly increased longevity over the Atm-/- mice, it appears that there may be two periods of tumor susceptibility: (a) the initial period up to 10 months where animals develop thymic lymphomas; and (b) for the survivors, a later onset of tumorigenesis with a distinct set of tumors. This second period of tumor susceptibility can be explained by the development of age-related tumors. No tumors were observed in wild-type controls during the same time frame. Thus, the phenotype of the mutant described here provides additional evidence for heterogeneity in A-T, and it provides a model to investigate additionally the molecular basis of tumor development in A-T.


   ACKNOWLEDGMENTS
 
We thank Dr. Glenda Gobe for helpful discussions, Dr. David Purdie for statistical analysis, Ella Harness and Margaret Yaakubowitz for expert technical help, and Ann Knight and Kylee Wallace for typing the manuscript. We also acknowledge the kind gift of Atm-/- from Dr. Philip Leder, Harvard University, Boston, MA.


   FOOTNOTES
 
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.

1 Supported by funds from the Australian National Health and Medical Research Council, the A-T Medical Research Foundation, and the A-T Children’s Project. Back

2 To whom requests for reprints should be addressed, at The Queensland Cancer Fund Research Unit, The Queensland Institute of Medical Research, P. O. Box Royal Brisbane Hospital, Herston, Brisbane, Queensland 4029, Australia. Phone: 617-3362-0341; Fax: 617-3362-0106; E-mail: martinL{at}qimr.edu.au Back

3 The abbreviations used are: A-T, ataxia-telangiectasia; TCR, T-cell receptor; mAb, monoclonal antibody; ISEL, in situ end-labeling; ES, embryonic stem; SP, single positive; DP, double positive; SPF, specific pathogen-free; FasL, Fas ligand; IL, interleukin. Back

4 I. Filippovich, unpublished observations. Back

Received 12/11/00. Accepted 4/ 2/01.


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 RESULTS
 DISCUSSION
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