Msh2 deficiency enhances somatic Apc and p53 mutations in Apc+/–Msh2–/– mice

Kyoung-Jin Sohn1, Monica Choi2, Jacquelin Song2, Sofeene Chan1, Alan Medline3, Steven Gallinger4,5 and Young-In Kim1,2,6,7

1 Department of Medicine,
2 Department of Nutritional Sciences,
3 Department of Laboratory Medicine and Pathobiology and
4 Department of Surgery, University of Toronto, Toronto, Ontario, M5S 1A8,
5 Samuel Lunenfeld Research Institute, Center for Cancer Genetics, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5 and
6 Division of Gastroenterology, Department of Medicine, St Michael’s Hospital, Toronto, Ontario, M5B 1W8, Canada


    Abstract
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 Abstract
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Inactivation of the adenomatous polyposis coli (Apc) gene by loss of the wild-type Apc allele (LOH) is a prerequisite for the development of intestinal adenomas in Msh2 proficient Min (Apc+/–Msh2+/+) mice. In contrast, adenomas from Msh2 deficient Min (Apc+/–Msh2–/–) mice are not usually associated with LOH. Given the role of Msh2 in post-replicative DNA repair, this study investigated whether Msh2 deficiency enhances somatic Apc and p53 mutations in Apc+/–Msh2–/– mice. Somatic Apc mutations (5/sample) were observed in the non-neoplastic intestinal mucosa from Apc+/–Msh2–/– mice but not from Min mice, suggesting that Msh2 deficiency is associated with a hypermutable state in the intestinal mucosa from Apc+/–Msh2–/– mice. Adenomas from Apc+/–Msh2–/– mice had a 2-fold higher rate of somatic Apc mutations (10/adenoma) than the non-neoplastic intestinal mucosa (5/sample), and did not demonstrate LOH. Truncating Apc mutations were observed in 82% of the adenomas from Apc+/–Msh2–/– mice and were not observed at all in the non-neoplastic intestinal mucosa. In contrast, in Min mice, all adenomas demonstrated LOH, had significantly less numbers of somatic Apc mutations (1.8 mutations/adenoma) compared with the adenomas from Apc+/–Msh2–/– mice, and harbored no truncating Apc mutations. These observations suggest that somatic Apc mutations, and not LOH, is a likely mechanism by which the Apc gene is inactivated in the development of adenomas in Apc+/–Msh2–/– mice in contrast to Min mice. Adenomas from Apc+/–Msh2–/– mice, but not from Min mice, also harbored somatic p53 mutations (mutation frequency of 45.5%), reflecting hypermutability associated with Msh2 deficiency. The nature and frequency of somatic Apc and p53 mutations in Apc+/–Msh2–/– mice suggest that many genomic sites, in addition to genes containing simple repeated sequences, are at risk of somatic mutations associated with Msh2 deficiency.

Abbreviations: Apc, adenomatous polyposis coli; CRC, colorectal cancer; LOH, loss of heterozygosity; MCR, mutation cluster region; MMR, mismatch repair; RER, replication error


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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The adenomatous polyposis coli (APC) tumor suppressor gene, termed the ‘gatekeeper’ of colorectal carcinogenesis, plays an important role in cell cycle regulation, apoptosis, cell adhesion and migration, microtubule assembly and signal transduction (1). Germline mutations of the APC gene lead to familial adenomatous polyposis (FAP), which is characterized by the development of hundreds to thousands of pre-malignant colorectal adenomas at a young age (1). In addition, somatic APC mutations have been observed in over 60% of sporadic colorectal cancer (CRC) (1). The majority of APC mutations are frameshifts or nonsense point mutations resulting in truncation of the APC protein (1). Min (multiple intestinal neoplasia; Apc+/–) mice, which carry a heterozygous germline mutation at codon 850 of the mouse Apc gene, develop ~25–75 small intestinal adenomas and one to five colorectal adenomas by 160–180 days of age, at which time they become moribund and die from anemia and intestinal obstruction (2,3).

Mismatch repair (MMR) genes ensure accurate replication of the genome during cell division (4,5). Mutations in the MMR genes result in a ‘mutator phenotype’, where loss of post-replicative DNA repair increases the mutation rate, and results in a replication error (RER) phenotype or microsatellite instability (4,5). Germline and somatic mutations in the MMR genes have been implicated in hereditary non-polyposis colorectal cancer syndrome (HNPCC) and in ~15–20% of sporadic CRC (4,5). Among several MMR genes identified and characterized thus far, the MSH2 and MLH1 genes have been observed to be most frequently mutated or silenced in both HNPCC and sporadic CRC (4,5). The contribution of the MSH2 gene in colorectal carcinogenesis has been supported by observations that Msh2 deficiency accelerates intestinal tumorigenesis in the Apc+/–Msh2–/– murine model (6). Apc+/–Msh2–/– mice, generated by crossing Min mice with mice carrying a homozygous mutation of the Msh2 gene (Msh2–/–), display an accelerated intestinal adenoma phenotype and develop numerous dysplastic colonic aberrant crypt foci (ACF), the probable earliest precursor of CRC (7), compared with Min mice (7). Apc+/–Msh2–/– mice develop ~350 small intestinal adenomas, eight colorectal adenomas and 55 ACF by 80 days of age, at which time they become moribund and die of anemia or bowel obstruction (6).

Inactivation of the Apc gene by loss of the wild-type Apc allele (LOH, loss of heterozygosity) is a prerequisite for intestinal tumor development in Min mice (8,9) as well as in Apc1638N mice, another Apc knockout murine model (10). In humans with FAP carrying a germline mutation in one APC allele, both somatic mutations and LOH have been observed in colorectal adenomas and cancers (8). In contrast, only 15% of adenomas from Apc+/–Msh2–/– mice demonstrate LOH despite the observation that these adenomas demonstrate absence of the Apc protein by immunostaining (6). Thus, mechanisms by which the Apc gene is inactivated in the development of the majority of intestinal adenomas in Apc+/–Msh2–/– mice are unknown at present. Because of the role of the MSH2 gene in post-replicative DNA repair (4,5), biallelic loss of Msh2 and resultant Msh2 deficiency may enhance somatic mutations of the wild-type Apc allele, leading to Apc inactivation in Apc+/–Msh2–/– mice. In support of this hypothesis, tissues of mice lacking Msh2 (Msh2–/–) and transgenic for a retrievable reporter gene have been observed to harbor strikingly increased spontaneous and chemically induced mutation frequencies compared with Msh2 heterozygotes (Msh2+/–) or wild-type mice (Msh2+/+) (1113). Furthermore, murine Msh2–/– thymic lymphomas have been shown to be associated with significantly elevated mutation frequencies compared with normal Msh2–/– tissues (14). Similar to Msh2–/– mice, mice lacking Pms2 (Pms2–/–), another MMR gene, and transgenic for a reporter gene have demonstrated elevated mutation frequencies in all tissues examined compared with Pms2+/– or Pms2+/+ mice (13,15). Also, human tumors and cell lines lacking MMR have demonstrated greatly elevated mutation rates of specific indicator genes compared with those proficient in MMR (1619).

This study investigated whether Msh2 deficiency enhances somatic Apc mutations within a 2738 base pair (bp) region in exon 15 of the Apc gene including the human mutation cluster region (MCR) in intestinal adenomas from Apc+/–Msh2–/– mice. The effect of Msh2 deficiency on somatic p53 mutations within a mutational hot spot for human CRC (exons 5–8) was also examined in order to determine whether the mutagenic effect associated with Msh2 deficiency is specific to the Apc gene or is a more widespread phenomenon.


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This study was approved by the Animal Care Committee of the Samuel Lunenfeld Research Institute.

Tissue samples
Eleven histologically confirmed intestinal adenomas from 11 Apc+/–Msh2–/– mice and eight from eight Min mice from two previously published studies (20,21) were analyzed in the present study. The detailed protocol of these studies has been published previously (20,21). Min mice were bred at the Samuel Lunenfeld Research Institute on the C57BL/6J strain (original breeding pair from The Jackson Laboratory, Bar Harbor, ME). Msh2–/– mice were generated by gene targeting (22). The generation of Apc+/–Msh2–/– mice from Min and Msh2–/– mice has been described previously (6). At the time of necropsy, all small intestinal polyps were identified and counted. Previous studies have shown that all small intestinal polyps are adenomas (6). Representative small intestinal polyps were harvested at the time of necropsy (11 and 15 weeks of age for Apc+/–Msh2–/– and Min mice, respectively), fixed in 10% buffered formalin and embedded in paraffin in a standard fashion. All polyps analyzed were of equivalent size. Five micrometer thick sections were cut and mounted on microscope slides for staining with H&E using standard techniques for histological confirmation.

DNA extraction
Areas corresponding to histologically confirmed adenomas on H&E staining were marked on matched unstained slides. DNA from each adenoma was extracted as crude preparations using proteinase K lysis mix (10 mM Tris–HCl, pH 8.0, 100 mM KCl, 2.5 mM MgCl2, 0.45% Tween 20 and 1 mg/ml proteinase K) as described previously (23,24). Care was taken to avoid contamination from adjacent non-neoplastic tissues. The sections were homogenized in the lysis mix and digested for 1 h at 65°C followed by 10 min at 95°C. Extracted DNA was stored at –20°C until subsequent analyses. DNA from the non-neoplastic small intestinal mucosa was extracted from areas corresponding to normal histology on H&E staining from matched unstained slides in a similar fashion (23,24).

Apc LOH assay
The PCR-based Apc locus quantification assay was performed as described by Luongo et al. (9). Briefly, following digestion with HindIII, the 144 bp PCR product from the allele carrying the Min mutation (ApcMin) allele and 123 bp product from the wild-type Apc (Apc+) allele were resolved on 6% polyacrylamide gels, followed by densitometric analysis. All samples were amplified in duplicate, and ratios of pairs differed by <10%. An Apc+ allele: ApcMin ratio of 0.85 is expected in normal tissue (no LOH) based on the difference in the number of radioactive deoxycytosine resides in the two alleles following digestion with HindIII.

Mutation analyses
Apc gene.
A 2738 bp region, between nucleotide (nt) 2020 and 4758 in exon 15 of the Apc gene, including a region designated as the MCR in human CRC (nt 3906–4589), was amplified by PCR using three pairs of exon primers to generate three overlapping segments (segment A, nt 2020–2996; segment B, nt 2863–3925; segment C, nt 3829–4758) as described previously (23,24). About 60% of the somatic mutations of the APC gene in human CRC are clustered in a 500 bp region in the MCR (25). The primer sequences were constructed based on the published murine Apc cDNA sequence (3) (GenBank accession no. M88127) and synthesized by the ACGT (Toronto, Ontario, Canada). The sequences of the primers were as follows: segment A: sense, 5'-ACACTCGAATTCAATCCTAAAGACCAGGAAGC-3'; antisense, 5'-ACACTCATCGATTGGCCTCTTTTACCATATCC-3'. Segment B: sense, 5'-TCTAGGTCTAGAAAACCCTCAGTTGAATCC-3'; antisense, 5'-ACACTCCTCGAGTGTTGTCTGATCACATCC-3'. Segment C: sense, 5'-TCTAGGGAATTCAACACAGGAAGCAGATTC-3'; antisense, 5'-ACACTCCTCGAGTCTACCTCTTTATCCTGG-3'.

Each 5.0 µl DNA sample was amplified by PCR in a 50 µl volume containing 350 ng of each primer, 0.25 mM each dNTP, PCR buffer (Qiagen, Mississauga, Ontario, Canada), 1.5 mM MgCl2 and 2 U HotStart Taq DNA polymerase (Qiagen). Following hot start PCR at 95°C for 5 min, 35 cycles of denaturation (95°C) for 1 min, annealing (54°C) for 1 min and extension (72°C) for 1 min were performed in a thermal cycler (PTC-200 DNA Engine; MJ Research, Watertown, MA). All PCR amplifications included a 10 min extension at 72°C after cycle 35.

p53 gene.
Exons 5–6 (366 bp) and 7–8 (576 bp) of the p53 gene were amplified by PCR using two pairs of intron primers. The majority of p53 mutations in human CRC occur within a highly conserved area spanning from codons 110 to 307 (exons 5–8) (26,27). The primer sequences were constructed based on the published murine p53 cDNA sequence (GenBank accession nos X01237 and K01700) (28) and synthesized by the ACGT Corp. The sequences of the primers were as follows: exons 5–6: sense, 5'-ACACTCGAATTCCTTCCAGTACTCTCCTCCCC-3'; antisense, 5'-TCTGTGCTCGAGAAGGTACCACCACGCTGTGG-3'. Exons 7–8: sense, 5'-GTGTCTGAATTCCCGGCTCTGAGTATACCACC-3'; antisense, 5'-GTGTCTCTCGAGGCCTGCGTACCTCTCTTTGC-3'.

Each 2.5 µl of DNA sample was amplified by PCR in a 50 µl volume containing 350 ng of each primer, 0.25 mM each dNTP, PCR buffer (Qiagen), 1.5 mM MgCl2 and 2 U of HotStart Taq DNA polymerase (Qiagen). Following hot start PCR at 95°C for 5 min, 35 cycles of denaturation (95°C) for 30 s, annealing (54°C) for 30 s and extension (72°C) for 45 s were performed in a thermal cycler (PTC-200 DNA Engine®; MJ Research). All PCR amplifications included a 10 min extension at 72°C after cycle 35.

Subcloning and sequencing.
The PCR products for the Apc (segments A–C) and p53 (exons 5–8) genes were gel purified using the Qiaex II Agarose Gel Extraction Kit (Qiagen) according to the manufacturer’s protocol, re-extracted and dissolved in 50 µl of double-distilled H2O. The PCR products were then subcloned into pBluescript II KS(+) vector (Stratagene, Cambridge, UK). Sequencing was performed on a total of two clones from each PCR product using the Dideoxy Terminator Label Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and an Applied Biosystems 373 Sequencer (Applied Biosystems) as described previously (23,24). The Apc and p53 sequences thus generated were analyzed against the GenBank sequences [Apc, accession no. M88127 (3); p53, accession nos X01237 and K01700 (28)] using the DNASIS software program (Hitachi, San Diego, CA). The allele specificity of Apc mutations was determined by the presence of the germline ApcMin mutation at codon 850 (TTG->TAG, leucine->stop). Mutations were confirmed by sequencing the opposite strand as well as sequencing one independent clone from each of three to five independently performed PCR reactions. In total, therefore, two clones from the initial PCR reaction and three to five independent clones from three to five separate PCR reactions were sequenced per tumor. Only those mutations consistently present in all of the sequencing analyses were considered to be real mutations. Non-neoplastic small intestinal mucosal DNA was PCR amplified under the same conditions for tumor DNA, and a total of two clones from each PCR reaction were sequenced initially, followed by sequencing one independent clone from a separately performed PCR reaction as described above.


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LOH in adenomas from Apc+/–Msh2–/– and Min mice
Consistent with previous observations (8,9), all adenomas from Min mice demonstrated LOH (Figure 1Go). In contrast, none of the 11 adenomas from Apc+/–Msh2–/– mice demonstrated LOH (Figure 1Go). Taken together, the data suggest that LOH is not a primary mechanism by which the Apc gene is inactivated in the development of intestinal adenomas in Apc+/–Msh2–/– mice.



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Fig. 1. LOH was observed in all intestinal adenomas from Min (Apc+/–Msh2+/+) mice but not in those from Apc+/–Msh2–/– mice. Shown are representative results of radioactive PCR-amplified Apc loci in DNA from normal small intestine (N) and intestinal adenomas (T) following digestion with HindIII. The 144 bp and 123 bp PCR products are from the ApcMin and Apc+ (wild-type) alleles, respectively.

 
Somatic Apc mutations in adenomas from Apc+/–Msh2–/– and Min mice
A total of 110 somatic mutations were found in the 2738 bp region in exon 15 of the Apc gene from all of the 11 adenomas from Apc+/–Msh2–/– mice (Tables I and IIGoGo). On average, each adenoma harbored 10 mutations. Seventy percent of the observed mutations (77 of 110) were missense point mutations resulting in an amino acid change (n = 62), and nonsense mutations (n = 3) and deletions (n = 12) resulting in truncation of the Apc protein (Tables I and IIGoGo). The remaining (30%) mutations were silent missense point mutations without an amino acid change (Tables I and IIGoGo). Nine of the 11 adenomas harbored truncating mutations (82%) (Tables I and IIGoGo).


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Table I. Summary of somatic p53 mutations in intestinal adenomas from Apc+/–Msh2–/– and Apc+/–Msh2+/+ (Min) mice

 

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Table II. Summary of somatic Apc mutations in intestinal adenomas from Apc+/–Msh2–/– and Apc+/–Msh2+/+ (Min) mice
 
The most common mutations were transitions (87 of 110 or 79%), of which A:T->G:C (38.2%) and T:A->C:G (20.0%) were most prevalent (Tables I and IIGoGo). Eleven (92%) of the observed 12 deletions were 1 bp deletions occurring within simple repeated sequences (two to five mononucleotide and dinucleotide repeats) whereas one deletion was within a non-repeat sequence (Tables I and IIGoGo). Only 20% (22 of 110) of the somatic Apc mutations were located within the region corresponding to the human MCR. Eighty percent (88 of 110) of the somatic Apc mutations were situated in the region spanning codons 702–1288 upstream of the MCR. No distinct mutational hot spots were identified. Both the wild-type Apc+ and ApcMin alleles harbored somatic mutations; the nature and frequency of somatic Apc mutations were similar between these alleles.

In contrast to somatic Apc mutations in adenomas, non-neoplastic small intestinal mucosal DNA from Apc+/–Msh2–/– mice harbored on average only five somatic Apc mutations in the same region. All mutations were transitions resulting in an amino acid change, 60% of which were C:G->T:A substitutions. No truncating or silent Apc mutations were observed in non-neoplastic small intestinal mucosal DNA in contrast to adenomas from these mice.

A total of 14 mutations found in the 2738 bp region in exon 15 of the Apc gene from all of the eight adenomas from Min mice (Tables I and IIGoGo). On average, each adenoma harbored 1.8 mutations. All mutations were missense point mutations resulting in an amino acid change, 71% of which were transitions (Tables I and IIGoGo). In contrast to adenomas from Apc+/–Msh2–/– mice, no truncating Apc mutations were observed in adenomas from Min mice (Tables I and IIGoGo). The most prevalent mutations were A:T->G:C (seven of 14 or 50%) transitions (Tables I and IIGoGo). Forty-three percent (six of 14) of the Apc mutations were located within the region corresponding to the human MCR. Both the wild-type Apc+ and ApcMin alleles harbored somatic mutations; the nature and frequency of somatic Apc mutations were similar between these alleles. No somatic Apc mutations were observed in non-neoplastic small intestinal mucosal DNA from Min mice.

Somatic p53 mutations in adenomas from Apc+/–Msh2–/– and Min mice
In order to determine whether somatic mutations observed in the Apc gene are limited to the Apc gene or represent a more widespread genomic hypermutability associated with Msh2 deficiency in Apc+/–Msh2–/– mice, somatic mutations of the mutational hot spot for human CRC in the p53 gene (exons 5–8) (26,27) were examined in the non-neoplastic small intestinal mucosa and adenomas from Apc+/–Msh2–/– mice. No somatic mutations were observed within exons 5–8 of the p53 gene in the non-neoplastic small intestinal mucosa from Apc+/–Msh2–/– mice. However, a total of six p53 mutations in exons 5–8 were found in five of the 11 adenomas analyzed (mutation frequency of 45.5%) (Tables III and IVGoGo). All mutations were missense point mutations resulting in an amino acid change, 83.3% of which were transitions (Tables III and IVGoGo). The most prevalent mutations were A:T->G:C (three of six or 50%) transitions (Tables III and IVGoGo). Although up to 50% of p53 mutations in human sporadic CRC are C:G->T:A transitions occurring at CpG dinucleotides within these exons (26,27), these mutations were not observed in intestinal adenomas from Apc+/–Msh2–/– mice. In contrast, no p53 mutations were observed in the non-neoplastic intestinal mucosa and adenomas from Min mice.


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Table III. Somatic p53 mutations in intestinal adenomas from Apc+/–Msh2–/– mice
 

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Table IV. Summary of somatic p53 mutations in intestinal adenomas from Apc+/–Msh2–/– and Apc+/–Msh2+/+ (Min) mice
 

    Discussion
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 Materials and methods
 Results
 Discussion
 References
 
The observation that somatic Apc mutations are present in the non-neoplastic small intestinal mucosa from Apc+/–Msh2–/– mice but not in that from Msh2 proficient Min mice suggests that Msh2 deficiency is associated with a hypermutable state in the non-neoplastic intestinal mucosa from Apc+/–Msh2–/– mice. The observation that intestinal adenomas from Apc+/–Msh2–/– mice have on average a 2-fold higher number of somatic Apc mutations than the non-neoplastic intestinal mucosa and are not associated with LOH suggests that somatic Apc mutations resulting from Msh2 deficiency, and not LOH, is a probable mechanism by which the Apc gene is inactivated in the development of adenomas in Apc+/–Msh2–/– mice. Truncating Apc mutations were observed in 82% of the adenomas from Apc+/–Msh2–/– mice and were not observed at all in the non-neoplastic intestinal mucosa. This observation suggests that inactivating Apc mutations are necessary for the development of adenomas in Apc+/–Msh2–/– mice. In contrast, all adenomas from Min mice demonstrated LOH and harbored no truncating Apc mutations, although somatic missense mutations (1.8/tumor) were observed at a rate much lower than that observed in adenomas (10/tumor) and non-neoplastic intestinal mucosa (5/tumor) from Apc+/–Msh2–/– mice. This suggests that the probable inactivating mechanism of the Apc gene in the development of adenomas in Msh2 proficient Min mice is LOH as described previously (8,9).

The truncating Apc mutation frequency (82%) and the lack of LOH observed in intestinal adenomas from Apc+/–Msh2–/– are consistent with observations made in other Apc knockout mice crossed with other MMR genes. For example, in Min mice crossed with Mlh1–/– mice, only 19% of 36 intestinal adenomas showed LOH while 21% of adenomas that failed to show LOH demonstrated truncating Apc mutations (29). In Apc1638N mice crossed with Mlh1–/– mice (Mlh1–/–Apc1638N), 84% of 44 intestinal adenomas demonstrated truncating Apc mutations within codons 677–1690 (30). In Apc1638N mice crossed with another type of Msh2–/– mice (Msh2{triangleup}7N/{triangleup}7N), no LOH was detected in intestinal adenomas; in contrast, truncating Apc mutations were found in 41% of 34 tumors examined (31). Our present study in conjuction with these studies suggest that somatic Apc mutations, and not LOH, are primarily responsible for the inactivation of the Apc gene in the development of intestinal adenomas in Apc knockout mice deficient in MMR function in contrast to LOH in Apc knockout mice proficient in MMR function.

One novel finding in this study is predominant missense Apc mutations (56% of all mutations) in adenomas from Apc+/–Msh2–/– mice. Previous animal studies only screened for truncating Apc mutations, and hence the frequency and nature of missense mutations were not determined (2931). Although the observed missense mutations result in an amino acid change, the functional ramifications of these mutations were not investigated in the present study. Only 24% of the observed missense mutations were located in the region corresponding to human MCR, whereas 76% were located in the region spanning codons 800–1288 upstream of the MCR. Because this region contains ß-catenin binding and down-regulation domains and Ser-Ala-Met-Pro motifs necessary for APC to bind to conductin and axin (31), the observed missense mutations in the present analysis may impair ß-catenin down-regulation by Apc and binding of Apc to conductin and axin. However, the following lines of evidence from the present study suggest that the majority of these missense Apc mutations are probably functionally neutral: (i) the non-neoplastic intestinal mucosa from Apc+/–Msh2–/– mice harbored on average five missense mutations; (ii) additional truncating Apc mutations appear to be a prerequisite for the development of adenomas from Apc+/–Msh2–/– mice (as evidenced by the truncating Apc mutation frequency of 82% in adenomas compared with 0% in the non-neoplastic intestinal mucosa); (iii) the frequency and nature of missense mutations were similar between the Apc+ and ApcMin alleles; and (iv) missense mutations were also found in adenomas from Min mice in which the wild-type Apc+ allele is lost. In the present analysis, 30% of all Apc mutations were silent. The functional ramifications of these silent mutations are unknown.

In the present study, 80% (12 of 15) of the observed truncating Apc mutations in adenomas from Apc+/–Msh2–/– mice were 1 bp deletions, all but one occurring within simple two to five mononucleotide and dinucleotide repeated sequences. This finding is consistent with observations from intestinal adenomas from other animal models of MMR deficiency (2931) and human RER+ CRC (32,33). In the present analysis, transitions were most prevalent mutations (79% of all mutations) in intestinal adenomas from Apc+/–Msh2–/– mice; A:T->G:C and T:A->C:G mutations accounted for 38 and 20% of all mutations, respectively. Frameshift mutations were observed in 11% of all mutations in intestinal adenomas from Apc+/–Msh2–/–. In the non-neoplastic intestinal mucosa from Apc+/–Msh2–/–, all mutations were transitions, 60% of which were C:G->T:A. In intestinal adenomas from other animal models of MMR deficiency and human RER+ CRC, the most prevalent somatic Apc point mutations are C:G->T:A transitions (3034). However, these studies only screened for truncating mutations, and hence the precise nature of point mutations cannot be ascertained (3034). Although 84% of point mutations observed in intestinal adenomas from Mlh1-/-Apc1638N mice were C:G->T:A transitions occurring at cytosine-guanine (CpG) dinucleotides (30), these mutations were not observed in intestinal adenomas from Apc+/–Msh2–/– mice. In normal small intestinal tissues from Msh2–/– mice that are transgenic for a reporter gene (lacI), the majority of spontaneous mutations were G:C->A:T transitions (42–48%) followed by A:T->G:C transitions (19–25%) (11,12). Frameshift mutations were less frequent with the mutation frequency of 14–19% in this model; these were predominantly –1 bp deletions (11,12). Thymic lymphomas arising from Msh2–/– mice also demonstrated predominantly G:C->A:T and A:T->G:C transitions (60%) followed by transversions (18%) and frameshifts (21%) in the lacI reporter gene (14). In normal small intestine tissue from Pms2–/– mice carrying the lacI reporter gene, the most common mutations were transitions (58%); however, these mice demonstrated a higher proportion of frameshift mutations (34 versus 23%) and a lower proportion of transversions (8 versus 13%) than Msh2–/– mice carrying the same reporter gene (13). These observations are consistent with the known functions of Msh2 and Pms2 proteins, which correct base substitution mismatches and slippage errors, respectively (13). Mutator strains of Escherichia coli deficient in MutS (MSH2 homolog) MMR functions display a mutator phenotype in which the mutations are predominantly transitions, the majority (>70%) of which are G:C->A:T and A:T->G:C transitions, and single nucleotide deletions (35,36). The predominance of transitions in the non-neoplastic intestinal mucosa and adenomas in Apc+/–Msh2–/– mice is in keeping with evidence from bacteria indicating a preference of proofreading activity of pol III for transversions over transitions (37).

In the present analysis, only 20% of the observed somatic Apc mutations were located within the region corresponding to the human MCR in intestinal adenomas from Apc+/–Msh2–/– mice. The remaining 80% of the observed Apc mutations in intestinal adenomas from Apc+/–Msh2–/– mice were located in the region spanning codons 702–1288 upstream of the MCR. No distinct mutational hot spots were identified in the present study. In intestinal adenomas from Mlh1–/–Apc1638N mice, all Apc mutations were observed between codons 791 and 1464, >50% of which were located upstream of codon 1019; codon 854 was a mutational hot spot for point mutations, while codons 927–929, 1209–1211 and 1461–1464 were mutational hot spots for frameshift mutations (30). In chemically induced CRC in rats, somatic Apc mutations (83–100%) have been observed to be clustered within a 757 bp region between codon 1026 and 1279 in exon 15 upstream of the MCR (23,38). In the present analysis, 35% (39 of 110) of all Apc mutations were located in this region, and 43% (47 of 110) were in a region spanning codons 702–1020. These regions contain ß-catenin binding and down-regulation domains and Ser-Ala-Met-Pro motifs necessary for APC to bind to conductin and axin (31). Therefore, in contrast to human colorectal carcinogenesis, distinct clusterings of mutations upstream of human MCR appear to exist in chemical carcinogen rodent models of CRC and MMR deficient Apc knockout murine models of intestinal tumorigenesis.

One surprising finding of the present study is the multiple number of Apc mutations per adenoma (mean = 10) in Apc+/–Msh2–/– mice. Some adenomas harbored multiple missense mutations even in the presence of truncating mutations. The high number of somatic Apc mutations observed in the non-neoplastic mucosa (mean = 5) in Apc+/–Msh2–/– mice is a novel finding that has not been reported previously in other Apc knockout mice crossed with other MMR genes (2931). We believe that the observed Apc mutations in Apc+/–Msh2–/– mice are real mutations for the following reasons: (i) all potential Apc mutations detected on initial screening were confirmed by sequencing three to five independent clones from three to five separately performed PCR reactions, and only those mutations consistently present in all of the confirmatory sequencing were reported; (ii) the analysis of p53 mutations from the same neoplastic tissues demonstrated a ‘normal’ degree of mutations; (iii) even if PCR and sequencing artifacts might have contributed to the unusually high number of Apc mutations detected in adenomas from Apc+/–Msh2–/– mice, the same amount and quality of neoplastic tissue from Min adenomas prepared and analyzed in the same manner harbored only 1.8 mutations/adenoma. Recently, CRC from the interleukin-2 and ß2-microglobulin deficient mouse, which is an animal model of ulcerative colitis-associated CRC and is associated with MMR deficiency, has been shown to harbor on average 6.1 somatic Apc mutations/tumor (24), suggesting that the Apc gene is particularly susceptible to the mutagenic effect of MMR deficiency. The unusually high number of Apc mutations would probably have come from multiple mutations on all alleles resulting from MMR deficiency. Although the occurrence of polyploidy has been observed in several human cancers as well as in murine tumors (3941), a recent study has shown an absence of generalized chromosomal instability in adenomas from Min mouse (42). This observation argues against polyploidy being a likely mechanism for increased Apc mutations in adenomas from Apc+/–Msh2–/– mice.

The predominance of somatic Apc point mutations and the occurrence of slippage of one nucleotide within simple two to five mononucleotide runs and dinucleotide repeated sequences in intestinal adenomas from Apc+/–Msh2–/– in the present analysis suggest that many other genes, in addition to those containing highly repetitive dinucleotide tracts, are potential targets for somatic mutations in MSH2 deficiency. The type II TGF-ß receptor gene which contains a mononucleotide (A)10 run and (GT)3 dinucleotide repeat (43) and the BAX gene which contains a mononucleotide (G)8 repeat (44) have been observed to be frequent targets for somatic frameshift mutations in RER+ human CRC and cell lines. Other genes, however, have also been observed to be targets for somatic mutations in MMR deficient CRC and cell lines. For instance, somatic ß-catenin mutations, which are more frequently found in RER+ human CRC than RER– CRC, are predominantly point mutations (mostly transitions) resulting in amino acid substitutions (4547). In the present study, we found that the p53 gene, another tumor suppressor gene frequently implicated in human colorectal carcinogenesis (26,27), is also a potential target for somatic mutations in intestinal tumorigenesis associated with Msh2 deficiency. In contrast to the somatic Apc mutations observed in the non-neoplastic intestinal mucosa, no somatic p53 mutations were observed in the non-neoplastic intestinal mucosa from Apc+/–Msh2–/– mice. This observation as well as the fact that a significantly higher frequency of somatic mutations was observed in the Apc gene compared with the p53 gene in intestinal adenomas from Apc+/–Msh2–/– mice suggest that the Apc gene may be at a higher risk of somatic mutations compared with the p53 gene in Msh2 deficiency. The observation that no p53 mutations were found in intestinal adenomas from Msh2 proficient Min mice suggests that Msh2 deficiency was probably responsible for somatic p53 mutations in intestinal adenomas from Apc+/–Msh2–/– mice. Whether the observed somatic p53 mutations are functionally significant or neutral in the development of adenomas in Apc+/–Msh2–/– mice was not determined in the present study. In humans, p53 mutations are observed at either a much lower or equal frequency in HNPCC and sporadic RER+ CRC than that observed in sporadic RER– CRC (4851).

In summary, Msh2 deficiency was associated with somatic Apc mutations in the non-neoplastic intestinal mucosa from Apc+/-Msh2-/- mice. Somatic Apc mutations were observed at a significantly higher rate in intestinal adenomas than in the non-neoplastic intestinal mucosa from Apc+/–Msh2–/– mice. This observation as well as the absence of LOH in intestinal adenomas from this model suggest that somatic truncating, and potentially functionally significant missense, Apc mutations, are a likely mechanism by which the Apc gene is inactivated in intestinal tumorigenesis in Apc+/–Msh2–/– mice. Msh2 deficiency was also associated with somatic p53 mutations in intestinal adenomas in this model. The nature and frequency of somatic Apc and p53 mutations in this model suggest that many genomic sites, in addition to genes containing simple repeated sequences, are at risk of somatic mutations associated with Msh2 deficiency.


    Notes
 
7 To whom correspondence should be addressed Email: youngin.kim{at}utoronto.ca Back


    Acknowledgments
 
We thank the staff of the Samuel Lunenfeld Research Institute Animal Colony for care of the mice used in this study. We thank Colleen Ash, Kazy Hay, and Hyeja Kim for technical support. Supported in part by grants from the American Institute for Cancer Research (to Y.-I.K. and S.G.), the Canadian Institutes of Health Research (to Y.-I.K.), the MRC/Canadian Association of Gastroenterology/BYK Canada (to Y.-I.K.), and the St. Michael’s Hospital Health Sciences Research Program (to Y.-I.K.). Y.-I.K. was supported in part by a scholarship from the Medical Research Council of Canada. Presented in part at the 2000 American Association for Cancer Research meeting, April 1–5, 2000, San Francisco, CA, USA, and published in abstract form in Proceedings of the American Association for Cancer Research 2000; 41: A536.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 30, 2002; revised October 15, 2002; accepted October 22, 2002.