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[Cancer Research 61, 7934-7942, November 1, 2001]
© 2001 American Association for Cancer Research

Molecular Biology and Genetics

The Distinct Spectra of Tumor-associated Apc Mutations in Mismatch Repair-deficient Apc1638N Mice Define the Roles of MSH3 and MSH6 in DNA Repair and Intestinal Tumorigenesis1

Mari Kuraguchi, Kan Yang, Edmund Wong, Elena Avdievich, Kunhua Fan, Richard D. Kolodner, Martin Lipkin, Anthony M. C. Brown, Raju Kucherlapati and Winfried Edelmann2

Strang Cancer Research Laboratory at The Rockefeller University, New York, New York 10021 [M. K., K. Y., K. F., M. L., A. M. C. B.]; Department of Cell Biology and Anatomy, Weill Medical College of Cornell University, New York, New York 10021 [A. M. C. B.]; Departments of Cell Biology [E. W., E. A., W. E.] and Molecular Genetics [R. K.], Albert Einstein College of Medicine, Bronx, New York 10461; and Ludwig Institute for Cancer Research, La Jolla, California 92093 [R. D. K.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammalian cells, mismatch recognition has been attributed to two partially redundant heterodimeric protein complexes of MutS homologues, MSH2-MSH3 and MSH2-MSH6. We have conducted a comparative analysis of Msh3 and Msh6 deficiency in mouse intestinal tumorigenesis by generating Apc1638N mice deficient in Msh3, Msh6 or both. We have found that Apc1638N mice defective in Msh6 show reduced survival and a 6–7-fold increase in intestinal tumor multiplicity. In contrast, Msh3-deficient Apc1638N mice showed no difference in survival and intestinal tumor multiplicity as compared with Apc1638N mice. However, when Msh3 deficiency is combined with Msh6 deficiency (Msh3-/-Msh6-/-Apc1638N), the survival rate of the mice was further reduced compared to Msh6-/-Apc1638N mice because of a high multiplicity of intestinal tumors at a younger age. Almost 90% of the intestinal tumors from both Msh6-/-Apc1638N and Msh3-/-Msh6-/-Apc1638N mice contained truncation mutations in the wild-type Apc allele. Apc mutations in Msh6-/-Apc1638N mice consisted predominantly of base substitutions (93%) creating stop codons, consistent with a major role for Msh6 in the repair of base-base mismatches. However, in Msh3-/-Msh6-/-Apc1638N tumors, we observed a mixture of base substitutions (46%) and frameshifts (54%), indicating that in Msh6-/-Apc1638N mice frameshift mutations in the Apc gene were suppressed by Msh3. Interestingly, all except one of the Apc mutations detected in mismatch repair-deficient intestinal tumors were located upstream of the third 20-amino acid ß-catenin binding repeat and before all of the Ser-Ala-Met-Pro repeats, suggesting that there is selection for loss of multiple domains involved in ß-catenin regulation. Our analysis therefore has revealed distinct mutational spectra and clarified the roles of Msh3 and Msh6 in DNA repair and intestinal tumorigenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA MMR3 is critical for maintaining genomic stability and multiple homologues of the bacterial methyl-directed MMR proteins, MutS and MutL, have been shown to be essential for MMR in humans (1, 2, 3) . Inherited mutations in MMR genes in humans are directly involved in the etiology of HNPCC, an autosomal dominant disorder that confers predisposition to colonic and other tumors, whereas somatic mutations in MMR genes underlie some sporadic cancers (4, 5, 6, 7, 8) . In human cells, mismatch recognition is attributed to two heterodimeric protein complexes of MutS homologues, MSH2-MSH3 and MSH2-MSH6 (9, 10, 11, 12) . Both of these complexes interact with a heterodimer of two MutL homologues, MLH1-PMS2. Previous studies in human cells have indicated that base-base mismatches are preferentially targeted by the MSH2-MSH6 complex. In contrast, MSH2-MSH6 and MSH2-MSH3 appear to be redundant in insertion/deletion loop repair with possibly some specificity of the latter complex for larger (4–5 bases) insertion/deletion loops (10 , 11 , 13, 14, 15, 16, 17) . Because loss of MSH2 inactivates the activity of both mismatch recognition heterodimers, it is understandable that MSH2 deficiency causes strong cancer predisposition in both mice (18, 19, 20) and humans (21 , 22) . Loss of MSH3 or MSH6 function alone causes a partial MMR defect, consistent with their roles in MMR (15 , 23) . This may explain the rarity of MSH6 and the absence of MSH3 germ-line mutations in typical HNPCC families (24, 25, 26) .

Germ-line mutations in the tumor suppressor gene APC lead to familial adenomatous polyposis, another autosomal dominant syndrome that imparts predisposition to colorectal cancer (27 , 28) . In familial adenomatous polyposis patients, mutation or loss of the wild-type APC allele is considered a rate-limiting step in tumor initiation. APC is also mutated in the majority of sporadic cases of colorectal cancers (4) and in a subset of HNPCC-derived tumors (29 , 30) . Wild-type APC encodes a 2843-amino acid protein, one of the normal functions of which is to facilitate the destabilization of ß-catenin, a protein involved in both cell adhesion and signal transduction (31 , 32) . APC acts as a component of the Axin or Conductin complex, which targets ß-catenin for degradation by the proteasome pathway (33 , 34) . The mechanism by which APC functions in this process is poorly understood, but the activity is localized to the central region of APC. This region contains a series of seven 20-amino acid ß-catenin binding repeats (35) and three SAMP repeats, which are binding sites for Axin (or Conductin; Refs. 33 , 34 ). In addition, recent reports suggest that APC also contains highly conserved NESs in this region, and that it shuttles ß-catenin from the nucleus and cytoplasm to a junctional compartment where the axin complex may be anchored (36 , 37) . Nearly all of the tumor-associated mutations in APC occur within the first 1500 codons, and approximately two-thirds of these somatic mutations are confined to a MCR located between codons 1286 and 1513 (38 , 39) . These tumor-associated mutations give rise to truncated APC proteins that retain one or two of the 20-amino acid repeats in addition to all three of the more NH2-terminal 15-amino acid ß-catenin binding sites. These truncated APC products are unable to down-regulate ß-catenin (31 , 35) . The inability to regulate ß-catenin concentrations in the cells results in excessive levels of cytosolic ß-catenin and entry of this protein into the nucleus, where it acts as a transcriptional coactivator of the DNA binding protein Tcf-4 (40, 41, 42) .

To clarify the role of the MMR proteins in vivo, we previously developed a series of mouse lines, each carrying an inactivating mutation in a different MMR gene. Mice carrying an Msh6 null mutation have a cancer predisposition phenotype, associated with a significantly reduced life span (43) . Msh3-/- mice develop tumors only late in life and do not show increased morbidity compared with wild-type animals (23) . When Msh6-/- and Msh3-/- mutations are combined, the tumor predisposition phenotype becomes indistinguishable from that caused by Msh2-/- or Mlh1-/- (23) . Although development of gastrointestinal tumors was observed in a subset of the Msh3-/-, Msh6-/-, and Msh3-/-Msh6-/- mice, the limited number of intestinal tumors in these mouse strains makes the assessment of the roles of Msh3 and Msh6 in intestinal tumorigenesis difficult. To circumvent this problem, we generated mouse lines that allow us to study the role of Msh3 and Msh6 in intestinal tumorigenesis by crossing the MMR-deficient mice with Apc1638N mice. Apc1638N mice spontaneously develop three to five intestinal tumors/animal within the first year of life (44) . Compared with other Apc mutant strains, such as ApcMin and Apc{Delta}716 (45 , 46) , Apc1638N mice display a milder cancer phenotype and are longer lived. This feature makes this mouse line an ideal model to study additional risk factors in intestinal tumorigenesis, such as MMR deficiency. Using this approach, we previously crossed the Apc1638N strain with Mlh1-/- and Msh2-/- mice and demonstrated that the presence of the Apc1638N germ-line mutation dramatically increased the multiplicity of intestinal tumors and reduced the age of onset. Furthermore, the analysis of tumor-associated Apc mutations from these mice revealed mutational signatures of Mlh1 and Msh2 deficiency (20 , 47 , 48) .

Here, using Msh3-/-, Msh6-/-, and Msh3-/-Msh6-/- mice that also carry the Apc1638N allele, we demonstrate that the loss of Msh6 alone is sufficient to cause a strong predisposition to intestinal tumors in Apc1638N mice. The additional loss of Msh3 in this strain further contributes to an accelerated rate of intestinal tumor formation and increased morbidity. These observed increases in tumor formation are attributable to a higher incidence of truncating mutations in the wild-type Apc allele. The distinct spectra of tumor-associated Apc mutations from Msh6-/- and Msh3-/-Msh6-/- mice delineate the specific and overlapping roles of Msh3 and Msh6 in MMR and illustrate how their loss, both individually and together, contributes to intestinal tumor development.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of MMR-deficient Apc1638N Mice.
For the generation of MMR-deficient Apc1638N mice, Msh3+/- and Msh6+/- mice were backcrossed four to six times to C57BL/6 and subsequently intercrossed with Apc1638N mice that were congenic for C57BL/6. Double heterozygous Msh3+/-Apc1638N or Msh6+/-Apc1638N mice were intercrossed to obtain Msh3-/-Apc1638N or Msh6-/-Apc1638N mice. To obtain triple mutant mice, males and females that were Msh3+/-Apc1638N and Msh6+/-Apc1638N were intercrossed. Mice that were heterozygous for all three genes were intercrossed to obtain Msh3-/-Msh6-/-Apc1638N mutant mice. The animals were genotyped by PCR-based methods (23 , 49) .

Analysis of Tumors.
After sacrifice, the GI tract was opened longitudinally and examined under a dissecting microscope for the presence of tumors. Tumors of the GI tract and other tissues were processed for paraffin embedding, and sections were prepared for H&E staining and immunohistochemistry. The expression of Apc protein expression in the GI tumors was studied by avidin-biotin-peroxidase technique using antibodies N-15 and C-20 directed against the NH2 or COOH terminus, respectively (Santa Cruz Biotechnology, Santa Cruz, CA). Statistical analyses of tumor incidence and number were performed using the Fisher exact probabilities and Mann-Whitney test or binominal calculation. Differences were considered significant with P < 0.05.

Preparation of Tumor DNA Samples for Molecular Analysis.
Thirteen intestinal tumors and four non-tumor tissue samples from Msh3-/-Apc1638N mice, 49 intestinal tumors and eight non-tumor tissue samples from Msh6-/-Apc1638N mice, and 40 intestinal tumors and 13 non-tumor tissue samples from Msh3-/-Msh6-/-Apc1638N mice were collected. In each case, intestinal tumors were dissected from the intestinal epithelium and immediately frozen in liquid nitrogen. Alternatively, tumors were resected out after sections of intestine were spread on paper and fixed overnight in 70% ethanol. Genomic DNA was prepared from tissue samples by overnight proteinase K digestion, followed by purification using a QIAamp Tissue kit (Qiagen).

Microsatellite Instability in Tumors.
Fifty ng of tumor DNA were used per PCR reaction. Cycling reactions were performed with end-labeled primers. For the analysis of the mononucleotide marker (U12235), a nested PCR strategy was used: 25 cycles of 94°C for 30 s, 60°C for 1 min, 72°C for 2 min for the first round of amplification, and 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s for the second round. Analysis of the dinucleotide markers (D1Mit36 and D7Mit91) was performed using a single round of amplification: 50 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s. PCR products were analyzed by electrophoresis on denaturing 6% polyacrylamide gels and exposure to Kodak BioMax autoradiographic film.

Analysis of Apc Truncation Mutations.
Codons 677-1690 of the mouse Apc gene were analyzed for truncation mutations by PCR and IVTT as described previously (48) but with some modifications. PCR amplification of the wild-type Apc allele was performed in two stages to eliminate coamplification of the inactivated Apc1638N allele using the forward primer 5'-TACAGCACTTGAAATCTCACAG-3' (nucleotides 1991–2012 of mouse Apc; GenBank accession no. M88127) and a wild-type allele-specific reverse primer 5'-GTTGTCATCCAGGTCTGGTG-3' (nucleotides 5123–5142). Fifty to 100 ng of genomic DNA from either frozen or 70% ethanol-fixed tumor and non-tumor tissue samples were amplified in 20-µl reactions containing Pfu DNA polymerase reaction buffer [20 mM Tris-HCl (pH 8.8), 2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100, and 0.1 mg/ml nuclease-free BSA] and 0.05 units/µl of Pfu turbo (Stratagene). Cycling conditions were one cycle of 94°C for 5 min, followed by 25 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 5 min, with one final extension cycle at 72°C for 5 min. This procedure resulted in amplification products of ~3.2 kb. Two overlapping segments of the Apc gene covering codons 677-1234 and 1100–1690 were subsequently amplified from aliquots of the first reactions using two pairs of PCR primers specific for IVTT. The primer sequences were as published (48) , except for the forward IVTT primer for the amplification of 1100–1690 fragment: 5'-GCGGATCCTAATACGACTCACTATAGGGAGACCACCATGGGTATGATGATGTATAGGTCAAGGGGAACCAGT-3'. This primer was modified to contain three extra methionine tags (underlined) prior to the Apc-specific sequence to enhance the detection of [35S]methionine-labeled translation products. PCR was performed in 30-µl reactions containing 1.2-µl aliquots of the first-stage reactions. Cycling conditions for both segments were as above except that 20 cycles were performed, and the annealing temperature was 57°C. After purification using a QIAquick PCR purification kit (Qiagen), 50–100 ng of purified PCR products were used as templates in 6 µl of IVTT reactions (TNT T7 Quick Coupled Transcription/Translation System; Promega Corp.) containing 2.5 µCi of [35S]methionine (Amersham). The reactions were incubated at 30°C for 1 h. Aliquots of the IVTT reactions were diluted 10-fold in SDS sample buffer, denatured at 100°C for 5 min, and then analyzed by 12% SDS-PAGE and fluorography. For characterization of tumor-specific mutations, the PCR products were digested with BamHI and HindIII, gel purified, and cloned into an SP64 vector. Individual clones were screened by IVTT to identify mutations, and their DNA sequences were determined.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Survival and Tumor Incidence in MMR-mutant Apc1638N Mice.
Apc1638N mice typically develop three to five intestinal tumors/animal in their first year of life (44) . We have shown previously that the introduction of the Apc1638N germ-line mutation into Mlh1-/- and Msh2-/- mice resulted in an increased intestinal tumor incidence, a dramatic increase in the intestinal tumor multiplicity per animal, and significantly lower survival (20 , 47) . To assess the effects of Msh3 and Msh6 deficiency in intestinal tumorigenesis, we mated Apc1638N mice with mice defective in either Msh3 or Msh6 or in both proteins and generated double- and triple-mutant mice. Similar to previous results with Msh2+/-Apc1638N and Mlh1+/-Apc1638N mutant mice, we did not observe a significant reduction in survival or an increase in tumor incidence or tumor multiplicity in Msh3+/-Apc1638N, Msh6+/-Apc1638N, or Msh3+/-Msh6+/-Apc1638N compound heterozygous mice up to 1 year of age as compared with Apc1638N mice. However, the presence of the mutant Apc allele in the homozygous mutant mice generally resulted in reduced survival, increased tumor incidence, and increased intestinal tumor multiplicity, particularly in the Msh6- and Msh3/Msh6-deficient backgrounds. This increase in tumor multiplicity was similar to that observed previously in Mlh1-/- and Msh2-/- mice, but the degree varied and depended on the type of MMR deficiency. There was no significant difference in survival between Msh3-deficient Apc1638N mice (n = 12) and Apc1638N mice (n = 18), which lived up to 13 and 14 months, respectively. However, Msh6- and Msh3/Msh6-deficient Apc1638N mice had significantly reduced life spans. Msh6-deficient Apc1638N mice (n = 18) did not survive beyond 6 months of age, whereas Msh3/Msh6-deficient Apc1638N mice (n = 10) all died by 3 months of age. The differences in survival are statistically significant between the Msh3/Msh6-deficient Apc1638N mice and the Msh6-deficient Apc1638N mice (P < 0.0001, log rank test), between the Msh3/Msh6-deficient Apc1638N mice and Msh3-deficient Apc1638N mice (P < 0.0001, log rank test), and between the Msh6-deficient Apc1638N and Msh3-deficient Apc1638N mice (P < 0.0001).

The reduced survival of animals with the mutant Apc allele is caused by an accelerated tumorigenesis in the GI tract of some of the MMR-deficient mouse lines. A comparison of the tumor incidence and tumor number in moribund mice of the different MMR lines is presented in Table 1Citation . Msh6-deficient Apc1638N mice developed 25.6 ± 9.2 intestinal tumors/animal. This tumor multiplicity was about 6–7-fold higher than that of Apc1638N mice (P < 0.0001). In contrast, Msh3-deficient Apc1638N mice showed only a small increase in the multiplicity of intestinal tumors compared with Apc1638N mice (5.1 ± 3.4 tumors versus 3.9 ± 3.1 tumors), which was not statistically significant. Msh3 deficiency in the Apc1638N mice on its own did not have a significant effect on either survival or intestinal tumor predisposition. However, when combined with Msh6 deficiency (Msh3-/-Msh6-/-Apc1638N), the triple-mutant mice developed an average of 39.4 ± 20.0 intestinal tumors, 54% higher than Msh6-/-Apc1638N mice. The increase in tumor multiplicity/mouse in the triple-mutant Msh3-/-Msh6-/-Apc1638N mice compared with the Msh6-/-Apc1638N mice did not reach statistical significance because of the small number of the triple-mutant mice available for analysis. However, it is important to note that the Msh3-/-Msh6-/-Apc1638N mutant mice developed tumors at a much younger age than the Msh6-/-Apc1638N animals (Table 1)Citation . Furthermore, the reduced survival and the increase in tumor number in the triple-mutant Msh3-/-Msh6-/-Apc1638N mice were comparable with those seen in Msh2-/-Apc1638N and Mlh1-/-Apc1638N mice (20 , 47) .


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  		Table 1 Tumor incidence and multiplicity of GI tumors in MMR-deficient Apc1638N mice

 
Distribution of Tumors and Histological Analysis.
The combination of the different MMR mutations with the Apc1638N allele restricted the tumor formation mostly to the GI tract. We observed only two extra-GI tumors. These were in the skin of an Msh3-/-Apc1638N and an Msh6-/-Apc1638N mouse (Table 2)Citation . Similar skin tumors have also been found in Msh2-, Msh3-, and Msh6-deficient mice (19 , 23) . We also assessed the distribution and histology of the GI tumors from the different MMR-deficient Apc1638N mice. Most of the tumors in these MMR-deficient Apc1638N mouse lines developed in the small intestine (duodenum, jejunum, and ileum), and the increased tumor multiplicity in Msh6-/-Apc1638N and Msh3-/-Msh6-/-Apc1638N mice is attributable to the high tumor number at these sites (Table 1)Citation . With the exception of an increase in tumor number in the colon of triple-mutant Msh3-/-Msh6-/-Apc1638N mice (P < 0.02), no significant increase was observed in the colon of the different mouse lines. It is of interest to note that the majority of GI tumors (18 of 34; 53%) had already progressed into malignant adenocarcinomas at a comparatively young age in the Msh6-/-Apc1638N mice (4.5 months), whereas in Apc1638N and Msh3-/-Apc1638N mice, only a smaller proportion of tumors [(7 of 17 (33%) and 11 of 27 (40%), respectively] had progressed to this late tumor stage at an increased age (9.5 and 9.3 months, respectively; Table 2Citation ). In the triple-mutant Msh3-/-Msh6-/-Apc1638N mice, only a small percentage of tumors progressed into adenocarcinomas (2 of 19; 10%), which may be attributable to the very young age at which these mice had to be analyzed.


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  		Table 2 Histological types of tumors in MMR-deficient Apc1638N mice

 
Microsatellite Instability and Loss of Apc in Tumors from MMR-deficient Apc1638N Mice.
The tumors in Msh2-/-, Msh3-/-, and Msh3-/-Msh6-/- mice characteristically exhibit a microsatellite instability phenotype similar to tumors derived from HNPCC patients (18 , 23 , 50) . To analyze this phenotype in the intestinal tumors of Msh3-/-, Msh6-/-, and Msh3-/-Msh6-/- mice on the Apc1638N background, we examined 9 tumors from Msh3-/-Apc1638N mice, 10 from Msh6-/-Apc1638N mice, and 10 from Msh3-/-Msh6-/-Apc1638N mice. We tested each of the tumors for instability at a mononucleotide repeat locus (U12235) and two dinucleotide repeat loci (D1Mit36 and D7Mit91). In all three loci, no microsatellite instability was observed in any of the tumors. These observations are consistent with results in Msh2-/-ApcMin tumors (51) . It is unclear why these tumors do not show microsatellite instability. However, it is possible that similar to the normal tissues from mismatch repair defective mice, it may only be possible to observe microsatellite instability in these tumor DNAs by performing the analysis at limiting dilutions to examine single DNA molecules for allelic differences. It is now well established that loss of Apc by somatic truncation mutation is associated with the increased GI tumorigenesis seen in Msh2-/- and Mlh1-/- mice that carry a mutant Apc allele. We also observed loss of full-length Apc protein expression in tumor cells from the mice used in this study (Fig. 1Citation and data not shown).



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  		Fig. 1. Intestinal tumor in Msh6-/-Apc1638N mice. A, solitary tumor in the proximal colon elevated from the mucosal surface (bar, 1.4 mm). B, immunohistochemical preparation of the same colonic tumor showed tubular adenoma features with reduction of APC protein expression in tumor cells, stained with anti-Apc antibody (bar, 185 µm).

 
Incidence of Apc Mutations in MMR-deficient Apc1638N Tumors.
To study the molecular mechanism of Apc loss in MMR-deficient Apc1638N tumors, we analyzed the distribution and characteristics of truncation mutations in the wild-type Apc using an IVTT assay. In view of prior mutational analysis in humans and mice, the region of Apc considered most likely to contain mutations was the first 3 kb of exon 15 (20 , 38 , 48 , 52) . A total of 49 tumor DNA samples from Msh6-/-Apc1638N and 40 tumor samples from Msh3-/-Msh6-/-Apc1638N mice were analyzed by IVTT, and truncated Apc products were detected in 44 of 49 (90%) and 35 of 40 (87%) tumors, respectively (Table 3)Citation . A large proportion of the tumor samples analyzed (49 and 63%, respectively) showed more than one novel-sized truncated polypeptide by IVTT. We have found previously that in such cases, the mutations are most likely derived either from polyclonal tumors or from two or more adjacent tumors that were not separated during dissection (48) . We also analyzed the available tumor DNA samples from Msh3-/-Apc1638N mice. Unlike in the other two MMR-defective strains, in this strain we detected Apc truncation mutations in only 6 of 13 (46%) tumor samples, an incidence similar to that observed in Apc1638N mice (3 of 10; 30%; Ref. 48 ). These results indicate that MMR deficiency, either by loss of Msh6 alone or in combination with Msh3, leads to a dramatic increase in tumor multiplicity in Apc1638N mice, and this is associated with an increased frequency of somatic truncation mutations in Apc.


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  		Table 3 Analysis of Apc truncation mutations in intestinal tumors from MMR-deficient Apc1638N mice

 
Spectrum of Apc Mutations in Msh6-/-Apc1638N Tumors.
To characterize the Msh6-/--associated mutations in Apc at the molecular level, the relevant PCR reaction products were cloned, identified by IVTT analysis, and sequenced. The 76 mutant sequences identified are shown in Table 4Citation , and the mutation spectrum is summarized in Table 5Citation . All but five Apc truncation mutations detected in Msh6-/-Apc1638N tumors were base substitutions (93%). The substitutions were predominantly C-T transitions, and 54 of 71 (76%) occurred at CpG dinucleotides within an arginine codon (Table 4)Citation . A particular hotspot, where 27 independent mutations (36% of total) were found, was localized at codon 854. This codon is preceded by a short tract of AG dinucleotide repeats (Table 4Citation , and the same mutation was also found in Mlh1-/-Apc1638N and Msh2-/-Apc1638N tumors (20 , 48) .


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  		Table 4 Sequence of Apc mutations in MMR-deficient Apc1638N tumors

 

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  		Table 5 Spectra of Apc truncation mutations in MMR-deficient Apc1638N tumors

 
Spectra of Apc Mutations in Msh3-/-Apc1638N and Msh3-/-Msh6-/-Apc1638N Tumors.
A total of seven mutations from six Msh3-/-Apc1638N intestinal tumors were similarly characterized. Of seven mutations, two were large deletions not associated with any repetitive sequences, three were 2-base deletion mutations, and two were base substitution mutations (Table 4)Citation . It should be noted that Msh3 deficiency alone did not significantly contribute to accelerated tumor predisposition in Apc1638N mice. Because the Msh3-/--derived intestinal tumors in Apc1638N mice did not show a much higher incidence of Apc truncation mutations than the tumors in Apc1638N mice, it would be difficult to distinguish the Msh3-/--mediated mutations from those that spontaneously occur in Apc1638N-derived tumors. However, the combination of Msh3 and Msh6 deficiency in Apc1638N mice led to a further reduction in survival and caused accelerated tumorigenesis as compared with Msh6-deficient Apc1638N mice. This increase in severity of the cancer phenotype is therefore attributable to loss of repair function mediated by Msh3. Similar to tumors in Msh6-/-Apc1638N mice, a majority of the tumors in the Msh3-/-Msh6-/-Apc1638N animals showed Apc truncation mutations, and we speculated that the intestinal tumors from the triple mutant mice would also harbor Apc mutational signatures specific to Msh3 deficiency, i.e. mainly frameshift mutations.

A total of 82 mutations from Msh3-/-Msh6-/-Apc1638N tumors were characterized (Table 4)Citation , and a mixture of base substitutions (46%) and frameshift mutations was observed (54%; Table 5Citation ). Most base substitution mutations found in this strain were the same as those found in Msh6-/-Apc1638N tumors. The predominant hotspot for base substitutions was again at codon 854 (16%), and 31 of 38 (81.5%) base substitutions occurred at CpG dinucleotides within arginine codons. These results indicate clearly that the mutational spectra for base substitutions in Msh6-/- and Msh3-/-Msh6-/--mediated tumors are similar. Unlike the Msh6-/-Apc1638N-derived tumors, frameshift mutations were observed frequently in Msh3-/-Msh6-/-Apc1638N tumors. This suggests that the intestinal tumor suppressor function of Msh3 in these circumstances results from the correction of frameshift mutations in Apc. The frameshift mutations observed occurred exclusively at short runs of mono- or dinucleotide repeats (Table 4)Citation . In Msh3-/-Msh6-/-Apc1638N mice, dinucleotide frameshifts accounted for 38% of all of the Apc mutations characterized. Most of these mutations (31 of 44 frameshifts) were within sequences consisting of four or five dinucleotide repeats with particularly striking hotspots at codons 927–929, 1209–1211, and 1461–1464 (Fig. 2)Citation . These mutational hotspots are in common with those found in Mlh1- and Msh2-deficient mice (20 , 48) . In addition, two dinucleotide deletions and two dinucleotide insertions were found at codons 852–853, comprising a short tract of AG dinucleotide repeats. A dinucleotide deletion and an insertion were also found at the same site in Msh3-/-Apc1638N and Msh6-/-Apc1638N-derived tumors, respectively, but none has been reported in tumors from Msh2-/-Apc1638N and Mlh1-/-Apc1638N mice (20 , 48) .



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  		Fig. 2. Distribution of Apc mutations. Diagram of Apc between codons 677 and 1690 showing positions and characteristics of truncation mutations detected in Msh6-/-Apc1638N (above) and Msh3-/-Msh6-/-Apc1638N (below) intestinal tumors. {blacktriangleup}, deletion; {blacktriangledown}, insertion; •, substitution. Each symbol represents an independent mutation. Note the common hotspots at positions 854 and 956 in tumors from both strains and additional hotspots at 929, 1211, and 1464 in Msh3-/-Msh6-/-Apc1638N-derived tumors. The three 15-amino acid, ; four 20-amino acid ß-catenin binding repeats, ; and one SAMP repeat, , in this segment of Apc are indicated. The two NESs are at the ends of the third and fourth 20-amino acid repeats.

 
Distribution of Apc Mutations.
The distribution of all of the Apc mutations identified in Msh6-/-Apc1638N and Msh3-/-Msh6-/-Apc1638N tumors is shown in Fig. 2Citation . All but one of the Apc mutations characterized were located upstream of the third 20-amino acid repeat ß-catenin binding site, starting at codon 1493, and all were located upstream of the first SAMP repeat, starting at codon 1562. Therefore, in this study the Apc mutations that led to the development of intestinal tumors caused the loss of at least five of seven 20-amino acid repeats and all of the Axin binding sites in the resulting mutant Apc proteins. There was also a difference in the distribution of Apc mutations between Msh6-/-Apc1638N and Msh3-/-Msh6-/-Apc1638N tumors. The majority of Msh6-/-Apc1638N-derived mutations (84%) was clustered upstream of codon 1019, resulting in loss of all of the known ß-catenin binding domains (Fig. 2)Citation . In contrast, the Apc mutations in Msh3-/-Msh6-/-Apc1638N tumors were more widely distributed over an ~3-kb region and largely consisted of frameshift mutations. The spectrum and distribution of the Apc mutations were thus dependent on the MMR genotypes of the tumors and were reflective of the specific type of MMR defect. In both types of MMR defect, however, all detected Apc mutations lay before the first SAMP repeat.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In mammalian cells, the initiation of DNA MMR is mediated by the MMR proteins MSH2, MSH3, MSH6, MLH1, and PMS2 (1, 2, 3) . Germ-line mutations in the genes that encode MSH2, MSH6, and MLH1 are associated with cancer susceptibility syndromes. In contrast, only a few examples of germ-line PMS2 mutations and no examples of germ-line mutations in MSH3 have been reported to be associated with cancer susceptibility syndromes (4, 5, 6, 7, 8) . To ascertain the roles of MSH3 and MSH6 in intestinal tumorigenesis, we generated and examined Apc1638N mice defective in Msh3, Msh6, and in both Msh3 and Msh6. Our results demonstrate that the repair activities of both Msh3 and Msh6 are important for the prevention of mutations that can lead to the initiation and progression of intestinal tumorigenesis.

In the absence of a predisposing Apc germ-line mutation, Msh3-/-, Msh6-/-, and Msh3-/-Msh6-/- mice develop gastrointestinal tumors at low to moderate incidence with tumors observed in 10, 38, and 75% of the animals, respectively (23) . The addition of the Apc1638N allele to the respective MMR-defective strains increased the intestinal tumor incidence to 100%, as reported previously for an Mlh1-deficient mouse strain (47) . There are, however, differences between the individual MMR genotypes with regard to survival and intestinal tumor multiplicity. Loss of Msh6 in Apc1638N mice resulted in a 6–7-fold increase in tumor multiplicity as compared with Apc1638N mice. Loss of Msh3 alone, however, showed a small increase in tumor multiplicity that was not statistically significant and had no effect on the survival of the Apc1638N mice. Msh3-/-Apc1638N mice survived as long as 13 months of age compared with 6 months for Msh6-/-Apc1638N mice, 2–3 months for Msh2-/-Apc1638N mice, and 4 months for Mlh1-/-Apc1638N mice (20 , 47) . The Msh3-/-Msh6-/- double-mutant Apc1638N mice had a severely reduced life span and displayed increased susceptibility to intestinal tumorigenesis similar to the Msh2-/-Apc1638N and Mlh1-/-Apc1638N mice (20 , 47) . Consistent with our previous findings with single mutant Msh3-/- and Msh6-/- animals (23) , the mouse models described here show that Msh6 is more critical to intestinal tumor suppression and survival than Msh3, even in the presence of the Apc1638N germ-line mutation. However, the fact that Msh6-/-Apc1638N mice have a longer life span and develop fewer tumors than Msh2-/-Apc1638N or Mlh1-/-Apc1638N mice (20 , 47) indicates a role for Msh3 in these processes. Such a role becomes even more evident in Msh3-/-Msh6-/-Apc1638N mice; the phenotype is almost indistinguishable from the phenotypes of Msh2-/-Apc1638N and Mlh1-/-Apc1638N mutant mice. These observations not only support the notion that there is some degree of functional redundancy between Msh3 and Msh6 but also suggest that Msh6 has a tumor suppressor function in intestinal tumorigenesis that cannot be fully compensated by Msh3 proficiency. Although Msh3 deficiency is significantly compensated for by Msh6 and alone does not play a significant role in GI tumorigenesis, it is possible that a large cohort of mice would reveal a more subtle role of Msh3 in this process.

Spontaneously arising intestinal tumors in Apc1638N mice lose the wild-type Apc allele, and in most cases this occurs through a mechanism involving the loss of the entire chromosome (53) . Previously, we demonstrated that an increased intestinal tumor multiplicity in Msh2-/-Apc1638N and Mlh1-/-Apc1638N mice was attributable to an increase in intragenic truncation mutations in the Apc tumor suppressor gene (20 , 48) . The Msh2 or Mlh1 deficiency caused a change in the prevailing mechanism of Apc inactivation from allelic loss of the Apc gene locus to somatic mutation of the Apc coding region in these tumors. Because the intestinal tumor multiplicity of Apc1638N mice was dramatically increased by Msh6 and Msh3/Msh6 deficiency, we were able to perform a comparative analysis of the mutational mechanism involved in Msh3 and Msh6 deficiency in vivo. As in Mlh1-deficient Apc1638N tumors, in nearly 90% of the intestinal tumor samples from Msh6-/-Apc1638N and Msh3-/-Msh6-/-Apc1638N mice, Apc was inactivated by intragenic protein-truncating mutations. In contrast, the incidence of Apc mutations in tumors from Msh3-/-Apc1638N was similar to that of Apc1638N mice. This result implies that the predominant mechanism of Apc inactivation is attributable to loss of the wild-type Apc allele as in Apc1638N mice and further supports the notion that Msh3 deficiency on its own does not have a significant effect on intestinal tumorigenesis.

Apc mutations in Msh6-/-Apc1638N tumors were predominantly base substitutions (93%) that created stop codons. This finding is consistent with the reported repair capabilities of Msh6-/- mouse cells, which are not capable of repairing base-base mismatches but efficiently repair insertion/deletion mismatches (43) . A high base substitution rate was also reported at the HPRT locus in the hMSH6-deficient human colon carcinoma cell line HCT-15 (54) and at the CAN1 locus in msh6 mutant yeast cells (55) .

The number of tumor-associated Apc mutations detected in Msh3-/-Apc1638N mice was limited because of the low intestinal tumor multiplicity in this mouse strain and the low incidence of Apc truncation mutations in these tumors. However, the overall similarity in the intestinal tumor predisposition between Msh3-/-Apc1638N mice and Apc1638N mice suggests that Msh6-dependent repair corrects most of the replication errors that occur in the Apc gene. This is consistent with experiments that show that it is possible to restore the capacity to repair base-base mismatches and most but not all insertion/deletion mutations to the hMSH3-/-/hMSH6-/- double-mutant HHUA cell line upon introduction of chromosome 2 (carrying hMSH2 and hMSH6; Ref. 15 ). A role of Msh3 in the repair of dinucleotide insertion/deletion mismatches is indicated by the binding of human (10) and yeast (56) MSH2-MSH3 complexes to such mismatches. Similarly, MSH2-MSH3 complexes have been shown to promote the repair of dinucleotide base insertion/deletion mismatches in human cell extracts and may play a role in the repair of such mismatches in the mouse because the repair of some dinucleotide insertion/deletion mismatches is defective in Msh3-/- embryonic stem cell extracts (16 , 23) . In the current study, seven Apc mutations were identified in Msh3-/- tumors. Five of these mutations consisted of frameshift mutations, of which two were large deletions unassociated with repetitive sequences and three were dinucleotide insertion/deletions that were not found in other MMR-deficient Apc1638N tumors (Table 4)Citation . These results suggest that possibly a small subset of insertion/deletion mispairs are corrected by Msh2-Msh3-mediated MMR. Similarly, the predominance of Apc base substitution mutations in Msh3-proficient Msh6-/- tumors implies that Msh3 may not participate in repair of base-base mismatches but rather plays a role in the repair of insertion/deletion mutations in vivo that is largely redundant with Msh6.

More definitive evidence for the role of Msh3 in MMR and intestinal tumorigenesis was obtained from the Apc mutational analysis of Msh3-/-Msh6-/-Apc1638N tumors. In Msh3-/-Msh6-/-Apc1638N tumors, 54% of the observed mutations were frameshifts, which were infrequent in Msh6-/-Apc1638N tumors, whereas the remainder were base substitutions. All of the frameshift mutations in Msh3-/-Msh6-/-Apc1638N intestinal tumors were insertion/deletions of one or two nucleotides that occurred at short runs of mono- or dinucleotide repeats, indicative of unrepaired replication slippage errors (Tables 4Citation and 5Citation ). This mutation spectrum is in good agreement with the reported inability of Msh3-/-Msh6-/- cell extracts to recognize or repair both base-base and unpaired dinucleotide mismatches (23 , 57) . Similar genetic instability and MMR deficiencies have also been observed in yeast and human cell lines that are mutant in both MSH3 and MSH6 (15 , 55) . It is noteworthy that the types of tumor-associated Apc mutations found in Msh3-/-Msh6-/-Apc1638N mice and those described in Msh2-/-Apc1638N and Mlh1-/-Apc1638N mice are similar (20 , 48) . The effect of Msh3 deficiency in Msh3-/-Msh6-/-Apc1638N mice is also evident from the difference in the distribution of Apc mutations. The majority of Msh6-/-Apc1638N-derived mutations (84%) were clustered upstream of all of the ß-catenin binding domains, whereas Msh3-/-Msh6-/-Apc1638N-derived mutations showed a fairly even spread throughout the 3-kb region, with 54% of the mutations clustered upstream of the first 15-amino acids ß-catenin binding repeat (Fig. 2)Citation . This change was clearly attributable to additional hotspots for frameshift mutations that were located in the more 3' region of Apc. The observed Apc mutations therefore show distinct mutational signatures that correspond to the respective MMR deficiency. Collectively, these data support the MMR model that base-base mismatches are primarily repaired by an MSH2-MSH6 complex, whereas MSH2-MSH6 and MSH2-MSH3 complexes are redundant in the repair of insertion/deletion mismatches (2) . They also illustrate a function of Msh3 in the Msh6-/-Apc1638N mice in the repair of frameshift mutations that contribute to intestinal tumorigenesis and imply that the Apc gene is a major target for such mutations in these mice.

All of the tumor-associated Apc mutations detected in Msh6-/-Apc1638N and Msh3-/-Msh6-/-Apc1638N mice were located between codons 780 and 1559, leading to premature termination of the Apc polypeptide. In human APC, the majority of somatic mutations in tumors are concentrated in the centrally localized MCR between codons 1286 and 1513 (38 , 39) . These mutations result in a truncated protein typically retaining one or two 20-amino acid repeats but lacking the remaining COOH-terminal structural motifs. Recently, it has been proposed that the APC protein also has a nuclear export function, shuttling ß-catenin from the nucleus and cytoplasm to the junctional compartment, where the Axin complex may be anchored (36) . Highly conserved NESs are located adjacent to the MCR in APC, i.e.. in the 3rd, 4th and 7th 20-amino acid repeats that are lost in APC mutant cancer cells (37) . Notably, all except one of the tumor-associated Apc mutations observed here were located upstream of the third 20-amino acid ß-catenin binding repeat. It is possible that selection of Apc mutations is primarily based on loss of its ability to facilitate ß-catenin regulation, which may require the simultaneous loss of multiple functional domains that include the 20-amino acid ß-catenin binding repeats, SAMP repeats, and NESs. In support of this model, all except one of the mutations identified in tumors from the MMR-deficient mice resulted in truncated polypeptides lacking all of the SAMP repeats, at least five of the 20-amino acid repeats, and all three 20-amino acid repeat-associated NESs (Fig. 2)Citation . It is notable that these mutations were detected exclusively in the formerly wild-type allele but not in the functionally defective Apc1638N allele. Collectively, these data suggest that protein-truncating mutations in the MCR of APC provide a selective advantage during tumor initiation because multiple functional domains that are necessary for ß-catenin regulation are eliminated.

In conclusion, loss of Msh6 but not Msh3 increases the multiplicity of intestinal tumors in Apc1638N mice. However, simultaneous loss of Msh3 and Msh6 further accelerates intestinal tumorigenesis. These increases are caused by a higher incidence of protein-truncating mutations in the wild-type Apc allele, allowing a comparative analysis of tumor-associated mutational signatures specifically attributable to either Msh3 or Msh6 deficiency in an in vivo mammalian system. The resulting data clearly indicate that base-base mismatch repair is dependent on Msh6, whereas Msh3 and Msh6 are redundant in the repair of some insertion/deletion mismatches. Finally, it is evident that the type and distribution of the somatic Apc mutations in MMR-deficient mice depend on the underlying mutator phenotype and are also selected for their inability to down-regulate ß-catenin.


   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 This work was supported by NIH Grants CA76329 (to W. E.), CA84301 (to R. K. and W. E.), CA29502 and CA47207 (to A. M. C. B.), CA67944 and N01-CN-65031 (to M. L. and R. K.), GM50006 (to R. D. K.), and Center Grant CA13330 to Albert Einstein College of Medicine; the AACR-Cancer Research Foundation of America Fellowship in Prevention Research (to M. K.); and an Irma T. Hirschl Career Scientist Award (to A. M. C. B.). Back

2 To whom requests for reprints should be addressed, at Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: (718) 430-2030; Fax: (718) 430-8574; E-mail: edelmann{at}aecom.yu.edu Back

3 The abbreviations used are: MMR, mismatch repair; APC, adenomatous polyposis coli; HNPCC, hereditary nonpolyposis colorectal cancer; SAMP, Ser-Ala-Met-Pro; NES, nuclear export signal; MCR, mutation cluster region; GI, gastrointestinal; IVTT, in vitro transcription and translation. Back

Received 6/11/01. Accepted 8/27/01.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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