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Carcinogenesis Advance Access originally published online on April 5, 2006
Carcinogenesis 2006 27(9):1860-1866; doi:10.1093/carcin/bgl029
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Most spontaneous tumors in a mouse model of Li-Fraumeni syndrome do not have a mutator phenotype

Kathleen A. Hill1,2, Victoria L. Buettner1, Analeah Heidt1,3, Lin-Ling Chen1, Wenyan Li1, Kelly D. Gonzalez1, Ji-Cheng Wang1, William A. Scaringe1,4 and Steve S. Sommer1,*

1 Department of Molecular Genetics, City of Hope/Beckman Research Institute Duarte, CA 91010, USA
2 Department of Biology, The University of Western Ontario London, Ontario, Canada N6A 5B7
4 Bioinformatics Group, Department of Molecular Genetics City of Hope, Duarte, CA 91010, USA
3 Present address: University of California San Francisco, CA, USA

*To whom correspondence should be addressed at: Department of Molecular Genetics, Beckman Research Institute/City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010-0269, USA. Tel: +1 626 930 5497; Fax: +1 626 301 8142; Email: sommerlab{at}coh.org


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutations are the substrate of cancer. Yet, little is known about the degree and nature of mutations in tumors because measurement of mutation load in tumors and normal tissues was generally not possible until the advent of transgenic mouse mutation detection systems. Herein, we present the first analysis of mutation frequency and pattern in thymic tumors from a mouse model of Li-Fraumeni syndrome (p53+/– murine model) using the Big Blue® assay with sequencing of all mutants. We also make the first characterization of mutation frequency and pattern in p53-deficient extra-thymic cancers. The data more than triple the literature on all non-mismatch repair deficient tumors for which mutations are identified by sequence analysis, allowing mutation frequency and pattern to be determined. Most tumors had a normal mutation frequency and a normal mutation pattern. Five tumors showed modest increases in mutation frequency (2.3-fold or less). Alterations in mutation patterns were uncommon, tumor-specific and not necessarily associated with increases in mutation frequency. Given the data from two spontaneous tumors (normal mutation frequency with an abnormal pattern in a p53–/– mouse and low mutation frequency in a p53+/+ control mouse), we hypothesize that tumors sometimes can carry a low mutation load. The study was not without certain caveats: mutation load could not be compared between tumor and normal tissue from the same animal; sample sizes for extra-thymic tumor types were small, and only point mutations and deletions, insertions and indels up to 2 kb were detected. However, the data clearly show key differences in tumors from p53+/– mice compared with mismatch repair deficient tumors; a lack of dramatic increase in mutation frequency and absence of a signature of mutation.

Abbreviations: MMR, mismatch repair; PCR, polymerase chain reaction


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Cancer is associated with an accumulation of somatic mutations of a magnitude hypothesized to result from a mutator phenotype (1–3), and certain cancers have demonstrated features of somatic mutation consistent with a mutator phenotype. DNA mismatch repair (MMR) deficiencies lead to dramatic increases in mutation frequency in normal tissues, predisposition to cancer and further dramatic enhancement of mutation frequency in the resulting tumors (4–6). Although features of a mutator phenotype are characterized and mutation load (i.e. mutation frequency and pattern) has been examined in MMR-deficient mouse models of tumorigenesis, little is known regarding mutation load in other spontaneous murine cancers. Elevations in mutant frequency were not observed in an analysis of four thymic lymphomas from p53–/– mice (7). The only complete characterization of mutation load in spontaneous tumors from non-MMR-deficient mice using a well-validated mutation detection system was an analysis of four thymic lymphomas from p53–/– mice (8). In that study, only one tumor had an elevated mutation frequency and that tumor had an altered mutation pattern.

Reduced p53 gene dosage in mice is associated with a spontaneous cancer phenotype with poor characterization of mutation load in tumors. Generally, mice deficient in p53 (i.e. gene knockouts with absence of p53 mRNA and p53 protein) appear to develop normally. All mice nullizygous for p53 (p53–/–) succumb to tumors, predominantly thymic lymphomas, by 10 months of age, with tumor development and death occurring as early as 3 months of age (9). Mice hemizygous for p53 (p53+/–) succumb to a more diverse panel of tumor types at a later age. More than 95% of p53+/– mice succumb to osteosarcomas, thymic lymphomas, splenic lymphomas and hemangiosarcomas by 2 years of age (9). Wild-type mice of the same genetic background show <20% mortality due to tumorigenesis by 2 years of age (10). In the p53+/– mouse, the p53 protein levels are reduced below that predicted on the basis of allele dosage (10) and the diversity of tumor types observed in p53+/– mice may reflect the susceptibility of individual tissues for the loss of the second p53 allele (11,12). Over 50% of the early-onset tumors (i.e. thymic lymphomas) and >85% of the late-onset tumors (i.e. >18 months and typically sarcomas) in this p53+/– mouse model retain the wild-type allele (10). Tumors that retain an intact p53 allele have been shown to express functional p53 protein, and in two cases the p53 transcript was sequenced and determined to be wild-type (10). Thus, p53-deficient mice provide an extensively studied phenotype of spontaneous tumorigenesis in which to characterize mutation load in individual, normal tissues and different tumor types with altered p53 gene dosage.

In an initial report, Sands et al. (7) observed similar mutant frequencies in four p53–/– thymic tumors and normal thymus tissue from p53–/– mice. In addition, Buettner et al. (8) sequenced Big Blue mutant plaques, allowing mutant and mutation frequencies and mutation pattern to be determined in four thymic lymphomas from p53–/– mice. One of four thymic lymphomas from p53–/– mice had an increased mutation frequency compared with normal thymus [TT108; 2.3-fold increase compared with normal thymus from p53–/– mice; (8)]. The mutation pattern in tumor TT108 showed a significant elevation in A:T to G:C transitions at non-CpG dinucleotides, distinguishing this pattern from that typically found in normal tissues, including thymus. Of the four tumors, TT108 was from the youngest mouse (2.5 months). Our preliminary results (8) led to a number of questions: (i) Will a larger sample of tumors confirm that most tumors have a normal mutation frequency and pattern? (ii) Is elevated mutation frequency in tumors associated with early tumor incidence? (iii) Is an increase in mutation frequency always associated with an altered mutation pattern? (iv) Is elevated mutation frequency, when it occurs, always associated with the same signature mutation pattern? (v) Does mutation load differ in tumors arising in animals with different p53 gene dosages? (vi) Will the results for thymic lymphomas, the most common tumor observed, be similar to the results obtained for other types of tumors? (vii) Can mutation frequency ever be reduced? To answer these questions mutation load was measured in additional tumors from p53–/–, p53+/– and p53+/+ mice. This is the first analysis of mutation load (i.e. mutation frequency and pattern) in thymic and extra-thymic tumors in a mouse model of the human Li-Fraumeni syndrome.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Animals and genotyping
Mice were housed in isolated air-filtered cages within a barrier facility and provided Purina 5058 laboratory diet (PMI Foods) and water ad libitum according to a protocol approved by the Institutional Animal Care and Use Committee. Histological reports were provided by Experimental Pathology Laboratories, EPL® (Herndon, VA). The protocols used herein were reported previously (13–17) (Stratagene Instruction Manual, August 15, 1992). Mouse genotypes were determined by polymerase chain reaction (PCR) for the wild-type p53 gene and by a combination of PCR and fluorescence in situ hybridization (FISH) for the lacI gene as described previously (14,15). The template for the PCR was genomic DNA isolated from tail. Loss of heterozygosity in tumors was assessed by robust dosage PCR (RD-PCR) as described previously (18–20). The primers for the wild-type p53 alleles (in exon 4) are as follows:

5'- GCGGTCCCAAAAGGGTCAGTACTTGGGCTTTGGTGTTGGG-3'
5'-GCGGTCCCAAAAGGGTCAGTTGAGGCACAGTCTACAGGCT-3'

The Big Blue® transgenic mouse mutation detection assay
Big Blue®/p53-deficient double transgenic mice provide a mouse model for dual analysis of mutation load and carcinogenesis (7). The Big Blue® transgenic mouse mutation detection system allows for the examination of spontaneous lacI transgene mutations in vivo in virtually any mouse tissue or cell type (13,17), including tumors (4–6). Big Blue mice harbor a chromosomally integrated lambda phage vector (LIZ) containing the E.coli lacI gene as the target of mutagenesis, which permits rescue of the lacI gene from mouse genomic DNA via in vitro packaging. Plating of packaged phage DNA in the presence of a chromogenic substrate (X-gal) provides a color screening assay in which mutant plaques give a blue phenotype and wild-type plaques are colorless. The entire lacI gene including the lacZ operator region can then be sequenced in these mutant plaques and mutations can be identified. The Big Blue lacI mutation screening assay is well validated (21–23) and spontaneous mutation frequencies are well defined for multiple tissues over the lifespan of the mouse (17,24,25). Mutation frequency in somatic tissues from Big Blue mice increases during gestation to young adulthood, is constant at ~2.5 x 10–5 in young to middle adulthood and increases in middle adulthood to old age in some tissues and not others. One pattern of mutation is found in all tissues examined with only minor tissue-specific differences observed with analysis of hundreds of mutations for detection (24). Similar findings are reported using the MutaMouse (i.e. lacZ mutation target) system (26). Herein, a total of 16 million plaque forming units (PFUs) were screened and mutations were identified in 1408 mutant plaques.

Measurement of mutation frequency and pattern
Mutation frequencies considered independent mutations only. Fold difference in mutation frequency was determined in comparison with control samples (i.e. genotype and age-matched, normal thymus, bone or spleen) examined simultaneously using the same lot of reagents in the standard block design (21). Mutation frequencies in the normal tissues were not significantly different from data reported previously for somatic tissues from animals 3–10 months of age (17,25). The mutation frequency in the hemangiosarcoma was not significantly different from that generally observed across somatic tissues at this age [2.5 x 10–5; (17,24,25)]. However, it was not possible to measure mutation frequency in a comparable normal tissue. Mutation patterns in tumors from p53-deficient mice were compared with control tissues examined simultaneously (i.e. genotype and aged-matched, normal thymus, spleen or bone).

Statistical analyses
Power analyses indicate that in the typical comparison of mutation frequencies in two tissue specimens, a 2.5 increase in mutation frequency would be detected with 80% power at significance level <0.05. Power analyses were performed using DSTPLAN (University of Texas M.D. Anderson Cancer Center, Department of Biomathematics, http://odin.mdacc.tmc.edu/) assuming a binomial distribution for the occurrence of an independent mutation, a null hypothesis that the mutation frequency is 2.5 x 10–5 in both tissue specimens and a two-sided test. Mutation frequencies were tested for significant differences using the Fisher's Exact Test 2 x 2 contingency tables (one-sided exact P-value; Cytel Software Corporation, Cambridge, MA). Mutation frequencies were compared with control samples (i.e. genotype and age-matched, normal thymus, bone or spleen) examined simultaneously using the same lot of reagents. Mutation frequency was not significantly different in normal tissues from two or three individual animals matched for p53 genotype and age. Mutation patterns were tested for significant differences by analysis as unordered r x c contingency tables using the ‘Fisher–Freeman–Halton’ test implemented by the StatXact statistical analysis software package (Cytel Software Corporation). For some observed r x c tables, the reference set is too large for practical computation of the exact P-value. In these cases, StatXact estimates the P-value and the corresponding 99% confidence interval from a sample of tables in the reference set, sampled in proportion to their probability (crude Monte Carlo sampling).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The questions raised by the initial study are addressed herein with an analysis of mutation load in a larger sample set of tumors from p53-deficient and wild-type mice. This study specifically looks for effects of p53 gene dosage, tumor type and p53-associated mutation signatures. Thirteen tumors and a second sample from TT108 are examined, including eight new thymic tumors and five new extra-thymic tumors (Table I). Six tumors from p53+/– mice were examined. These occur much later than in p53 nullizygous mice and extra-thymic tumors are much more frequent. Three thymic and three extra-thymic tumors from p53+/– mice as well as six tumors from p53 nullizygous mice (Table I) were analyzed. One out of six tumors from p53+/– mice show an elevated mutation frequency (Table I). Absolute mutation frequencies were similar in thymus and the extra-thymic tissues. Mutation frequencies in extra-thymic tumors are not significantly different from that observed in normal tissues. Spontaneous mutation frequencies and patterns from p53+/– tumors are compared with those in normal thymus, bone and spleen tissues of age- and genotype-matched control mice.


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  		Table I Mutation frequency is unchanged or moderately increased in tumors from p53-deficient mice and reduced in one tumor from a wild-type mouse

 

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  		Table II The pattern of mutation is altered in 3 of 13 tumors in p53-deficient mice

 
Extra-thymic tumors from p53+/– mice show an average 0.84-fold elevation in mutation frequency compared with normal tissue from mice matched for age and p53 genotype, whereas thymic lymphomas show a 1.5-fold elevation on average.

A minority of tumors show only a modest elevation in mutation frequency in both p53+/– and p53–/– mice. There is no obvious relationship between age and elevated mutation frequency; the thymic lymphomas from p53–/– mice with elevated mutation frequency occur in animals 2.5 to 5 months of age. The average age of p53–/– animals with elevated versus unchanged mutation frequency in thymic tumors is 3.4 and 5.5 months, respectively.

Increases in mutation frequency are not always associated with alterations in mutation pattern. Only three tumors have altered mutation patterns and the tumors were thymic lymphomas (Table II). Note that TT-86 and TT-108 both had elevated mutation frequencies and altered patterns. There is no characteristic mutation signature in tumors from p53-deficient mice since the three tumors have significantly different mutation patterns from each other (P = 0.01; Fisher's Exact Test 3 x 11). Tumor TT108 has an altered pattern of mutation with an over-representation of A:T to G:C transitions. TT86 has an over-abundance of T:A to G:C transversions (P = 0.02) and an absence of deletions/insertions in 30 independent mutations identified (P = 0.01). Tumor TT401 has an over-representation of A:T to G:C mutations (P = 0.02). Mutation patterns are similar among the other tumors despite alterations in mutation frequency in some tumors and constancy in others (Figure 1).


Figure 1
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  		Fig. 1 Summary of the alterations in mutation load observed in 16 spontaneous tumors from p53-deficient mice.

 
The spontaneous splenic lymphoma SL216 harvested from a p53+/+ mouse shows significantly reduced mutation frequency (0.5-fold; P = 0.005) from that in normal spleen from wild-type and p53+/– mice (Table I; also see statistical methods).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Examination of 16 tumors from p53-deficient mice reveals that (i) modest increases in spontaneous mutation frequency are observed in a minority of tumors in p53-deficient mice; (ii) elevated mutation frequency does not appear to be associated with earlier tumor occurrence; (iii) alterations in mutation pattern are observed in only a few tumors (three thymic tumors) and altered mutation frequency need not be associated with alterations in mutation pattern; (iv) there is no single signature alteration in the pattern of spontaneous mutations characteristic of p53-deficient tumors; (v) mutation load is generally similar in tumors from p53+/– and p53–/– mice (Figure 1); in addition, reduced mutation frequency was observed in a spontaneous tumor from a wild-type mouse. The data are not consistent with the hypothesis that tumors generally have a mutator phenotype.

A plasmid-based selectable system has been used to examine mutant frequency in tumors from p53–/– and p53+/+ mice and reported higher mutant frequencies in tumors versus normal liver and spleen tissue (27). However, individual mutations were not reported, only a subset of mutants was sequenced, mutation frequency was not determined and a detailed mutation pattern is not available for comparison with the data presented herein. Data collected with a plasmid-based system are difficult to interpret given the previously documented differences in mutation data compared with phage-based systems. In brief, the plasmid-based lacZ system generated elevated mutation frequencies in seminiferous tubules, similar to frequencies observed in somatic tissues (28). This is significantly different from the Big Blue lacI phage screening assay (17,29). Mutation patterns generated with the plasmid-based system show tissue and age specificity (30–32) but mutation patterns generated using phage-based assays do not (17,25,26,33). In addition, the ‘mutation fingerprints of aging’ reported using the lacZ-plasmid selectable system are not observed using the identical transgene in a phage-based selectable system (26). Thus, mutation patterns observed using a plasmid-based system (28,30–32) show unexplained and uninvestigated differences from the well-validated phage-based systems, even from a phage-based system using the same transgene (26). The lacI screening system used herein has been validated using seven lines of evidence (23), and selectable plasmid-based systems should be validated in a similar manner.

The observation of reduced mutation frequency in a tumor presents an interesting possibility that requires additional study. How can mutation frequency be decreased in a tumor? Also, how is it that mutation frequency plateaus in young to mid-adulthood (17,25) where time from conception to young increases 3-fold? How is it that certain tissues like sperm and neurons maintain a constant mutation frequency into old age (17,24–26). These observations might be explained by mutation frequency maintained in a steady state owing to a balance between mutation and cell proliferation and apoptosis (25). Increased levels of apoptosis in a tumor could shift the balance and result in lower mutation load. If occasional rogue cells with repair deficiencies carry most of the mutations within the tissue (25), it is possible for tumors to have a lower mutation frequency in at least one of two ways: (i) the rogue cells that form during the normal process of mutation from the cancer clone are more likely to apoptose, changing the balance, reducing the fraction of rogue cells relative to normal cells, or (ii) by chance, the initial rogue cells produced by somatic mutation-induced deficiency in repair occurs one or a few cycles later than expected, resulting in a 2- to 4-fold lower fraction of rogue cells versus normally mutating cells. We hypothesize that tumor SL-216 arose from a repair proficient cell and had a lower mutation frequency by one of the above mechanisms and tumor TT-401 arose in the same way and then acquired a mild repair deficiency, explaining how a tumor could have a normal mutation frequency, yet an altered pattern of mutation. Clearly, additional analyses of tumors are necessary to confirm that indeed mutation frequency can be reduced in spontaneous tumors. The rat Big Blue model may be preferred, as the larger size of the tissue allows mutation load to be assayed in tumor and normal tissue from the same animal.

Proposed functions of the p53 protein integral to DNA repair, apoptosis and cell proliferation predict the potential for increased mutation load and even a mutator phenotype in p53-deficient tissues. p53 has been proposed as the ‘guardian of the genome’ (34), and despite a great deal of study, the function of p53 is complex and not completely understood [reviewed in ref. (35)]. p53 is mutated in a wide variety of human cancers (36,37). Human lymphoblastoid cells with reduced p53 function showed elevated spontaneous and induced mutant frequency as measured at the thymidine kinase locus (38). Increased mutations resulting from non-homologous recombination were also observed for the adenine phosphoribosyl transferase (APRT) gene (39). Like human cells with p53 deficiency, p53-deficient mice have defective G2 (40), and mitotic spindle checkpoint control (41) resulting in numerical chromosome changes including both aneuploidy and polyploidy. These multiple lines of evidence indicate that loss of p53 function has the effect of reducing genomic integrity in multiple ways with the potential for the creation of a mutator phenotype.

Several lines of evidence suggested that p53 is not the ‘guardian of the genome’ in the general form of the hypothesis. Loss of p53 function did not necessarily elevate point mutations or alter mutation pattern (see below). In addition, two mouse minisatellites, previously found to be mutable after exposure to carcinogenic agents, did not show genomic instability in p53-knockout embryos (42,43). Also, mutation rates at two expanded simple tandem repeat loci in the male germline were similar in p53+/–, p53–/– and isogenic wild-type (p53+/+) mice (44). Increased p53 levels were found in somatic cells in response to agents associated with recombinogenic DNA double-strand breaks, yet no increase in meiotic recombination events was detected in an analysis of eight DNA markers on four different chromosomes in p53–/– mice (45). The role of p53 in the induction of apoptosis is also more complex than envisioned in the paradigm described above. On the one hand, certain cells such as thymocytes appear to be resistant to DNA damage-induced apoptosis when p53 function is lost (46). On the other hand, proliferating lymphocytes and lymphoma T cells remain sensitive to radiation-induced apoptosis even when p53 is deleted, suggesting that p53-independent apoptotic pathways might be activated when the p53 pathway is disabled (47). These data do not provide evidence for an association between p53 deficiency and spontaneous hypermutation or increased susceptibility to induced mutagenesis.

Previously, we observed a similar spontaneous mutation frequency and pattern in normal tissues (i.e. brain, spleen, liver and thymus) of p53+/– and p53 –/– Big Blue® transgenic mice (8,15,48). These data were consistent with a similar and independent analysis of normal thymus tissue from p53–/– and p53+/+ mice (7). Reduced p53 gene dosage did not appear to alter either spontaneous or induced mutant frequency measured using lacI, lacZ and APRT mutation targets and 10 different tissues/cell types (reviewed in ref. 35). We reported elevated doublet frequencies (i.e. 3-fold elevation in two non-tandem mutations identified in a single mutant) in normal tissues (liver and spleen) but not tumors from p53+/– mice (49). With few exceptions, the data indicate that reduced p53 gene dosage in normal mouse tissues is not associated with elevated spontaneous mutation load or increased sensitivity to mutagen exposure contrary to predictions of the ‘guardian of the genome’ hypothesis.

An important caveat in the present study is that the mutation frequency in a tumor is compared with that in a normal tissue specimen from a different animal (a control matched for age and genotype). The tumors in thymus and spleen tissues overwhelmed the normal tissue, so it was not possible to obtain a normal tissue specimen from the same animal. However, analyzing mutation rather than mutant frequency eliminates much of the inter-animal variation observed in mutant frequencies (21) since sequencing allows removal of jackpot mutations (i.e. mutations observed more than once owing to proliferation after the mutational event), which are responsible for much of the inter-animal variation (50). This caveat notwithstanding, the two tumors with increases in mutation frequency and altered mutation pattern are likely to reflect the biological event that alters both the frequency and nature of mutation. It is clear that the tumors in p53-deficient mice have little or no increase in mutation frequency, in contrast to the tumors in MMR-deficient mice in which there is an ~250-fold increase in mutation frequency (6).

Along with our previous pilot study, this is the first characterization of mutation load in animals without MMR deficiency. The p53-deficient mouse model of carcinogenesis is characterized by frequent tumors despite no general elevation of mutation load in normal tissues and infrequent, modest elevation in mutation load in tumors. These observations are not consistent with the general hypotheses that p53 is the ‘guardian of the genome’ and that tumors generally have a mutator phenotype. The data suggest that p53-deficient tumors generally have not acquired an MMR deficiency or other type of repair defect that might predispose to enhance development of chemotherapeutic resistance on one hand and/or increase apoptosis on the other hand owing to an increased mutation burden. The observations made herein are directly relevant for ~30% of Li-Fraumeni syndrome cases that have protein truncating mutations (Sommer, unpublished data). Also, the data hint that p53 mutagenesis, which occurs in 50% of human solid tumors, is not associated with defects in repair pathways.


Figure 2
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  		Fig. 2 Key observations regarding mutation load and altered p53 gene dosage in spontaneous tumors.

 


   Acknowledgments
 
We thank Drs Frisk and Weiss for examining tissue preparations and identifying the tumor tissues. We thank Asanga Halangoda for his help in the data collection. We also thank Hagen Blazyk and Petra Heinmoller for review of the manuscript and Christine Kim and Gayle Copeland for analyzing data. We thank Dr Kai Li for helpful discussion and careful review of the manuscript. The work is supported in part by RO1 NS33354.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 16, 2005; revised March 21, 2006; accepted March 22, 2006.


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J. Wang, K. D. Gonzalez, W. A. Scaringe, K. Tsai, N. Liu, D. Gu, W. Li, K. A. Hill, and S. S. Sommer
From the Cover: Evidence for mutation showers
PNAS, May 15, 2007; 104(20): 8403 - 8408.
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