The presence of increased frequencies of blood-derived and solid tumors in ataxia-telangiectasia (A-T) patients, coupled with a role for the ATM (A-T mutation) protein in detecting specific forms of DNA damage, has led to the assumption of a mutator phenotype in ATM-deficient cells. Supporting this assumption are observations of increased rates of chromosomal aberrations and intrachromosomal homologous recombinational events in the cells of A-T patients. We have bred mice with knockout mutations for the selectable Aprt (adenine phosphoribosyltransferase) locus and the Atm locus to examine the frequency of second-step autosomal mutations in Atm-deficient cells. Two solid tissues were examined: (a) the ear, which yields predominately mesenchymal cells; and (b) the kidney, which yields predominately epithelial cells. We report here the lack of a mutator phenotype for inactivating autosomal mutations in solid tissues of the Atm-deficient mice.

A-T5 is a rare autosomal recessive disorder associated with a greater than 60-fold increased risk of developing blood-derived or solid tissue cancers. Approximately 75% of these cancers are lymphomas and leukemias, with the remainder being distributed to a variety of solid tissues (1). The increased rate of cancer in A-T patients is believed to result, at least in part, from chromosomal instability and increased mutant frequencies that have been reported in their cells (2). However, only a small number of mutational studies have been performed to date, and studies of cells taken directly from patients, as opposed to studies with established cell lines, have been limited to blood-derived cells (3, 4, 5). There have also been reports that A-T carriers (i.e., ATM heterozygotes) have a 3–5-fold increased risk for solid tumors (6). This increase could result from an elevated sensitivity to genotoxins that cause double-strand breaks (e.g., ionizing radiation; Ref. 6), from increased rates of mutation in those cells suffering a second-step mutation at the wild-type ATM locus (7), or a combination of both possibilities. Detailed information on ATM status in tumors derived from heterozygous deficient individuals is still lacking, as is information about mutant frequencies for ATM homozygous deficient cells that are obtained from solid tissues. Such data are important because the reported increased risk of cancer in ATM carriers is still controversial (8, 9, 10). We have combined the murine Atm knockout mutation with a knockout mutation for the selectable Aprt locus (11) to determine in vivo mutant frequencies at Aprt in solid tissues removed from mice with heterozygous or homozygous deficiencies at Atm. We report here the lack of a mutator phenotype for inactivating autosomal mutations in solid tissues of the Atm-deficient mice.

Generation of Mice with Atm Homozygous and Heterozygous Deficiencies.

Atm homozygous deficient animals were generated by breeding Aprt heterozygous C57Bl/6 males with Atm heterozygous DBA/2 females and identifying F1 animals with double heterozygous deficiencies. Genotyping for Aprt(11) and Atm(12) was performed as described elsewhere. These F1 animals were then backcrossed with Atm heterozygous DBA/2 animals. Five Atm heterozygous, seven Atm homozygous, and one wild-type animal obtained from these backcrosses were used for experiments. The remaining animals were obtained from the original breedings used to create the F1 animals.

Determination of Mutant Frequencies for Ear and Kidney Tissues.

The mutational assay relies on Aprt as a reporter of mutational activity and is based on determining the relative percentage of homozygous deficient cells in enzymatically digested tissues removed from mice that are heterozygous at Aprt. The methods for generating kidney (13) and ear (11) cell suspensions are reported elsewhere. Aprt-deficient cells are identified by plating the cell suspensions into culture medium containing the adenine analogue DAP, which is toxic to cells with adenine phosphoribosyltransferase enzymatic activity. The mutant frequencies are calculated by dividing the number of DAP-resistant clones by the total number of clonable units plated. This latter number is determined with cloning efficiency plates containing medium without DAP.

Molecular Characterization of Mutational Events.

The methods used and the microsatellite loci examined are reported elsewhere (14).

Two solid tissues were examined: (a) the ear, which yields predominately mesenchymal cells (11); and (b) the kidney, which yields predominately epithelial cells (13). The results for ear and kidney tissues obtained from Atm heterozygous (n = 9) and homozygous deficient (n = 7) mice and from wild-type mice (n = 9) are shown in Fig. 1. With the exception of two wild-type mice, the two ears and two kidneys from each animal were examined separately, yielding four data points/animal. Most Atm homozygous animals had multiple tumors when sacrificed. A wide variation in mutant frequencies was noted for ear and kidney tissues, with a mean of approximately 10−4 for all three genotypes and for both organs (Table 1). The detection of very high mutant frequencies in some organs (Fig. 1) demonstrates that the Aprt mutation assay can detect elevated mutant frequencies. Moreover, with one exception, no more than one of four tissue samples from a given mouse had a markedly elevated mutant frequency. This exception was an Atm heterozygous mouse that yielded two kidneys with mutant frequencies near 1 × 10−2. These kidneys were excluded from further analysis because DAP-resistant clones selected from these kidneys failed to grow upon subculture. Data from one additional kidney from a heterozygous animal was not available for the analysis. A statistical analysis using logistic regression failed to demonstrate an increase in mutant frequencies for ear or kidney tissues removed from either the Atm heterozygous or homozygous animals. When the data points were separated by tissues and by sides, the Ps ranged from 0.16–0.76. The Ps were 0.42 for the kidney and 0.32 for the ear when both sides of each animal were pooled for each tissue examined.

The absence of an increase in the mutant cell frequency did not preclude the possibility that mutations formed via different mechanisms in Atm heterozygous and/or homozygous cells. Specifically, it has been suggested in work examining HPRT mutant T cells isolated from A-T patients that deletional events occur preferentially in ATM homozygous human cells (3). A limited number of DAP-resistant clones from each of the three genotypes were examined with chromosome 8 polymorphic microsatellite loci (14). Each mutational event was placed into one of three categories: (a) intragenic events (i.e., bp substitution, small deletion or insertion, epigenetic change); (b) interstitial deletion; or (c) mitotic recombination/chromosome loss (Table 2). Recombinational events could not be unambiguously distinguished from chromosome loss in all mutant cells because many of the animals had significant homozygosity for microsatellite loci between Aprt and the centromere due to meiotic recombination (see “Materials and Methods” for a description of the crosses used to generate the animals). However, all animals maintained heterozygosity for microsatellite loci between Aprt and the telomere, which is approximately 10 cM distal to Aprt(14), and for at least two microsatellite loci proximal to Aprt. For all three genotypes, the predominant mutational events were correlated with LOH for Aprt, and most (75–83% of the total) were placed in the mitotic recombination/chromosome loss group. A previous study has shown that mitotic recombination is the predominant mutational event in vivo, at least for wild-type animals (15). Deletional events were not found to be the predominant mutational event for either of the Atm genotypes, although the combined percentage of deletional events (16%, 5 of 32 events) is higher than that observed for the wild-type cells (5.5%, 1 of 18 events). Larger numbers of mutational events will need to be analyzed to determine whether this result is significant.

It has been suggested that cells from A-T patients are under chronic oxidative stress, which may account for part of the A-T phenotype (16). At the DNA level, oxidative damage is mutagenic, and we have shown that it causes a novel mutational pattern characterized by discontinuous LOH when Aprt is the target (14). No examples of discontinuous LOH were observed for mutant cells derived from Atm homozygous animals, and only one example, for a single microsatellite marker, was observed for mutant cells derived from heterozygous animals. Although these results suggest that oxidative damage did not play a significant role in causing mutations in the Atm-deficient mice, the lack of heterozygosity over the entire length of chromosome 8 suggests that this tentative conclusion should be interpreted cautiously at this time.

As mentioned in the “Introduction,” it has been suggested that A-T carriers are at risk for cancer if some of their cells undergo a second-step mutation at the ATM locus (7). To determine whether mutations in Atm heterozygous animals could be enhanced in cells converted to Atm homozygous, 16 Aprt homozygous deficient clones obtained from Aprt/Atm heterozygous deficient animals were examined for loss of the wild-type Atm allele. All 16 retained the wild-type allele, providing further evidence against enhanced mutagenesis in Atm homozygous deficient cells.

The Aprt system is particularly useful for detecting the broad spectrum of second-step mutations that can inactivate tumor suppressor genes (14), and it can also detect epigenetic inactivation (17). Therefore, the results obtained suggest that the increased rate of solid tumor formation in A-T patients is not due to an elevated rate of mutation. However, two caveats are noted. One is that the mice being studied were housed in clean rooms and were not exposed to external genotoxins, whereas humans are exposed continuously to a wide variety of exogenous genotoxins. We are currently using cultured cell lines to test the possibly that Atm homozygous and/or heterozygous deficient cell lines will exhibit a hypermutagenic response to ionizing radiation. The second caveat is that the Aprt assay does not detect readily translocation or intragenic homologous recombinational events, which may play significant roles in the formation of blood-derived cancers. Both events have been reported to be elevated in cells from A-T patients (2). Moreover, a recently completed study using the pink-eyed unstable (pun) mouse mutation to measure intragenic recombinational events demonstrated an increase in Atm homozygous deficient animals.6 It has also been reported that cultured embryonic fibroblasts from Atm homozygous deficient animals exhibit increased levels of chromosomal breaks (18). Nonetheless, the results obtained in this study demonstrate clearly that a spontaneous mutator phenotype for inactivating autosomal mutations is not present in the solid tissues of Atm heterozygous and homozygous animals. Further work will be necessary to determine whether other types of mutations or other pathways will account for solid tumor formation in A-T patients.

Fig. 1.

Aprt mutant frequencies in kidneys (▵) and ears (○) removed from wild-type (wt) mice, and Atm heterozygous (+/−) and homozygous (−/−) mice. Each symbol represents the mutant frequency for a single tissue, with the exception of those designated with an asterisk, which represent pooled kidney or ear from both sides of a single animal. Closed symbols represent cases where no mutant was identified, and the highest possible mutant frequency is shown.

Fig. 1.

Aprt mutant frequencies in kidneys (▵) and ears (○) removed from wild-type (wt) mice, and Atm heterozygous (+/−) and homozygous (−/−) mice. Each symbol represents the mutant frequency for a single tissue, with the exception of those designated with an asterisk, which represent pooled kidney or ear from both sides of a single animal. Closed symbols represent cases where no mutant was identified, and the highest possible mutant frequency is shown.

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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 NIH Grants CA56383 (to M. S. T.), PO 1 ES05652 (to P. J. S.) and DK38185 (to J. A. T.).

5

The abbreviations used are: A-T, ataxia-telangiectasia; DAP, 2,6-diaminopurine; LOH, loss of heterozygosity.

6

A. J. R. Bishop, C. Barlow, A. J. Wynshaw-Boris, and R. H. Scheistl. ATM deficiency causes an increased frequency of intrachromosomal recombination in mice, submitted for publication.

Table 1

Mutant frequencies by tissue and genotype

Atm genotypeOrganNo. of clones/tissueaMutant frequencyb
+/+ Kidney 22,700 (2,198) 0.9 (0.2) 
+/+ Ear 37,729 (4,430) 1.1 (0.2) 
+/− Kidney 13,855 (1,149) 0.8 (0.3) 
+/− Ear 68,146 (14,841) 2.1 (0.7) 
−/− Kidney 10,846 (1,495) 2.1 (1.3) 
−/− Ear 35,882 (5,600) 1.1 (0.4) 
Atm genotypeOrganNo. of clones/tissueaMutant frequencyb
+/+ Kidney 22,700 (2,198) 0.9 (0.2) 
+/+ Ear 37,729 (4,430) 1.1 (0.2) 
+/− Kidney 13,855 (1,149) 0.8 (0.3) 
+/− Ear 68,146 (14,841) 2.1 (0.7) 
−/− Kidney 10,846 (1,495) 2.1 (1.3) 
−/− Ear 35,882 (5,600) 1.1 (0.4) 
a

The average number of clonable units obtained for each tissue per genotype. The number in parentheses indicates the SE.

b

The average mutant frequency (×10−4) for each tissue per genotype is shown. Frequency determinations were made using all data collected, including those from tissues that yielded no DAP-resistant clones (see Fig. 1 legend). The number in parentheses indicates the SE.

Table 2

Mutational spectraa by genotype

Atm genotypenIntragenic eventsbDeletionscRecombination/Chromosome lossd
+/+ 18 15 
+/− 16 12 
−/− 16 13 
Atm genotypenIntragenic eventsbDeletionscRecombination/Chromosome lossd
+/+ 18 15 
+/− 16 12 
−/− 16 13 
a

The polymorphic microsatellite loci used for the mutational analysis are described elsewhere (14).

b

Intragenic events were defined by retention of the wild-type appearing Aprt allele. These events can include bp substitution, small insertions or deletions, and epigenetic change.

c

Deletions were defined by LOH for Aprt and retention of heterozygosity for centromeric and telomeric microsatellite loci.

d

Recombination/chromosome loss was defined by LOH for Aprt and all telomeric markers.

We thank Michael J. Lasarev for help with the statistical analysis of the mutant frequency data.

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