Heterozygous DNA mismatch repair gene PMS2-knockout mice are susceptible to intestinal tumor induction with N-methyl-N-nitrosourea
Xiusheng Qin,
Darryl Shibata1 and
Stanton L. Gerson2
Division of Hematology/Oncology and Ireland Cancer Center, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, OH 44106-4937 and
1 Department of Pathology, University of Southern California School of Medicine, Los Angeles, CA 90033, USA
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Abstract
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PMS2-deficient (PMS2/) mice are hypersensitive to N-methyl-N-nitrosourea (MNU)-induced thymic lymphomas based on the failure to initiate mismatch repair (MMR) at O6-methylguanine:T mismatches formed after MNU exposure. However, heterozygous PMS2 knockout (PMS2+/) mice do not develop spontaneous tumors, suggesting that they have sufficient MMR function to prevent genomic instability. We hypothesized that in PMS2+/ mice, exogenous carcinogens may either mutationally knockout the remaining normal allele leading cells to develop tumors or introduce sufficient DNA adducts and mismatches to overload the lower capacity for MMR, leading in either case to an increased rate of tumor production. In the present study, PMS2+/ mice and their littermate PMS2+/+ mice were monitored for tumor incidence following MNU treatment. Mice were given 50 mg MNU/kg i.p. when 5 weeks old. They demonstrated a similar incidence of thymic lymphomas, suggesting that expression of the single normal PMS2 allele is sufficient to protect the thymus and implying that a single dose of MNU may not efficiently knock out the remaining PMS2 allele in the thymus. Surprisingly, PMS2+/ mice were significantly more likely to develop intestinal tumorsboth adenomas and adenocarcinomasafter MNU than were PMS2+/+ mice (2.34 ± 0.34 tumors per mouse versus 1.34 ± 0.25 tumors per mouse; P < 0.05). The intestinal tumors were located mainly in the small intestine. However, these tumors in both the PMS2+/ mice and PMS2+/+ mice did not show microsatellite instability characteristic of loss of MMR. These results suggest that a single normal PMS2 allele can protect thymus but not intestine from MNU carcinogenesis. Organ-specific factors might influence MMR- mediated resistance to methylating agents. Heterozygous PMS2 knockout mice may be used as a promising animal model for intestinal tumorigenesis studies involving environmental carcinogens.
Abbreviations: APC, adenomatous polyposis coli; GI, gastrointestinal; HNPCC, hereditary non-polyposis colorectal cancer; MMR, mismatch repair; MNU, N-methyl-N-nitrosourea; MSI, microsatellite instability; O6-meG, O6-methylguanine.
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Introduction
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DNA mismatch repair (MMR) plays an important role in the prevention of tumorigenesis (1). In humans, defective mismatch repair system is causative of hereditary non-polyposis colorectal cancer (HNPCC), which comprises 513% of all colon cancers (1,2). Loss of DNA MMR is a rate-limiting step in the etiology of tumors associated with HNPCC (3). Affected HNPCC family members have inherited a mutant allele in one of the MMR-related genes, MSH2, MLH1, PMS1 or PMS2 (1). In tumors, the wild-type allele is lost through mutational inactivation, promotor methylation or loss of heterozygosity (LOH) (47). Mice defective in the mismatch repair gene, MSH2 or MLH1, are susceptible to intestinal tumors as well as thymic lymphomas and skin tumors (813). Moreover, these mice were reported to show accelerated adenomatous polyposis coli (APC) gene mediated intestinal tumorigenesis (13,14). However, deficiency in PMS2 does not predispose mice to intestinal tumors during the first 17 months of life (10). Recent studies have demonstrated that homozygous deficiency for PMS2 predisposed Min mice, heterozygous for the APC gene, to develop approximately three times the number of intestinal adenomas and four times the number of colon adenomas relative to Min and PMS2+//Min mice. However, there was no evidence of progression to carcinoma in these adenomas (15).
To understand and further interfere with the tumorigenesis in HNPCC patients, heterozygous mice should closely resemble the condition of HNPCC patients. However, mice heterozygous for PMS2 (PMS2+/) and PMS2+//Min mice failed to show an increased rate of tumor formation compared with PMS2+/+ and Min mice, respectively (15). Similarly, MSH2+/ and MSH2+//Min mice also did not demonstrate accelerated APC-mediated intestinal tumorigenesis (14), although 40% more extra-intestinal tumors were seen in the older MSH2+/ mice than in the control group of wild-type animals (16). Recently it was reported that 14% of the heterozygous MLH1 knockout mice (MLH1+/) developed gastrointestinal (GI) tumors, and that the GI tumor incidence increased 7-fold when the Apc1638N mutant was also present (13).
In the present study, the effect of N-methyl-N-nitrosourea (MNU) on PMS2+/ mice was investigated. PMS2 knockout mice were generated in 129 ES cells and chimeric mice were propagated as heterozygous mice in the C57BL/6 background (10). The genotypes of all the mice were determined by PCR amplification of tail DNA. Primers used were P1 (5'-TTCGGTGACAGATTTGTAAATG-3'), P2 (5'-TTTACGGAGCCCTGGC-3') and P3 (5'-TCACCATAAAAATAGTTTCCCG-3'), resulting in an ~300 bp product for the wild-type allele and an ~180 bp product diagnostic of a targeted allele as reported previously (10). Except for mice in the control groups, all experimental mice received a single i.p. injection of MNU, 50 mg/kg body wt, at 5 weeks of age. Mice were carefully observed for 1 year. Mice that developed thymic lymphoma typically had respiratory distress, and were killed under carbon dioxide (CO2) anesthesia and autopsied, and an immediate necropsy was done on all the dead mice. At autopsy, thymic lymphomas were massive, encasing the heart and the lungs with splenomegaly and hepatomegaly in some cases. Intensive invasion into many other organs including liver, lung, kidney, lymph nodes and brain were found in some of the mice. The tumors were intermediate and large non-cleaved cells, consistent with a lymphoblastic lymphoma. The small intestines and the colons were longitudinally cut open and fixed in 10% buffered formalin after they were inflated and washed with saline. All tumor-like lesions were recorded and later confirmed by histopathological examination. All other tissues were also fixed in 10% buffered formalin and were routinely processed for light microscopy examination. The sections were cut at 3 µm and stained with hematoxylin and eosin (H&E). Statistical significance was assessed by the
2 test and Fisher's exact test.
A total of 115 MNU-treated PMS2+/- mice, 73 MNU-treated PMS2+/+ mice, and 49 untreated PMS2+/- mice and 35 untreated PMS2+/+ mice were observed in the present study. There were no significant differences in overall survival between PMS2+/ and PMS2+/+ mice, both in the treated and untreated groups, as shown in Figure 1
. The PMS2+/ mice showed a significantly higher incidence of intestinal tumors including both adenomas and adenocarcinomas if they survived >20 weeks after treatment (Figure 2
), and had higher tumor multiplicity (2.34 ± 0.34 tumors per mouse in PMS+/ mice versus 1.34 ± 0.25 tumors per mouse in PMS2+/+, P < 0.05; Table I
). Most of the intestinal tumors were located in the small intestine as a small depressed or ulcerous lesions (Table I
; Figure 3
), which were difficult to distinguish from lymph follicles without histopathological examination. The histopathology demonstrated that most of the lesions were non-invasive adenomas and some of them were adenocarcinomas with aggressive invasion (Table I
; Figure 4
).

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Fig. 1. KaplanMeier survival curves. The data from 35 untreated PMS2+/+ mice (the top line) and 49 untreated PMS2+/ mice (triangles) were plotted for natural survival. Additionally, the data from 73 MNU-treated PMS2+/+ mice (squares) and 115 MNU-treated PMS2+/ mice (circles) were plotted to evaluate the effect of MNU on survival. There was no significant differences in overall survival between PMS2+/ and PMS2+/+ mice, both in the untreated and treated groups.
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Fig. 2. Intestinal tumor development in MNU treated mice. Mice were autopsied as indicated in Table I . The number of GI tumor bearing mice compared with the total number of mice autopsied for each time interval (<20, 2030, 3040 and >40 weeks old) is shown. The bar graph indicates the incidence of tumors observed during each time internal. The PMS2+/ mice showed a significantly higher incidence of intestinal tumors (P < 0.05).
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Fig. 3. Histology of intestinal adenoma induced by MNU. Histology of a representative adenoma of the small intestine of a mouse treated with MNU is shown (H&E staining; original magnification: 10x).
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Fig. 4. Histology of intestinal adenocarcinoma induced by MNU. Histology of a representative adenocarcinoma of the small intestine of a mouse treated with MNU is shown. This tumor obviously demonstrates its invasive characteristic (H&E staining; original magnification: 10x).
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No spontaneous thymic lymphomas were found in the untreated control PMS2+/ and PMS2+/+ mice (data not shown). Following MNU treatment, PMS2+/ and PMS2+/+ mice demonstrated a similar incidence of thymic lymphoma development, as shown in Figure 5
.

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Fig. 5. KaplanMeier survival curves for MNU-induced thymic lymphoma development in PMS2+/+ (squares) and PMS2+/ mice (circles). There was no significant difference between the groups. No spontaneous thymic lymphomas were found in the untreated control PMS2+/ and PMS2+/+ mice (data not shown).
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Mismatch repair (MMR) protein complex stabilizes the cellular genome by correcting single mismatches and single and dinucleotide slippage during replication. The protein complex recognizes the mismatch and recruits endonuclease, forming a long single-strand patch in the newly synthesized strand. The patch is then filled by polymerase
and
and sealed by ligase (1). Mice defective in PMS2 develop spontaneous lymphomas and sarcomas, but not intestinal tumors (10). PMS2+/ mice develop normally with no predisposition to any tumors. Here we confirmed that expression of the single normal PMS2 allele is sufficient to protect the T cells in the thymus from spontaneous and MNU-induced tumors, indicating that a single dose of MNU does not mutate the remaining PMS2 allele in the thymus, and thus an induced PMS2-deficient state does not occur. Interestingly, PMS2+/ mice were significantly more likely to develop intestinal tumorsboth adenomas and adenocarcinomasafter MNU than were PMS2+/+ mice.
Since PMS2+/ mice do not develop spontaneous thymic lymphomas and intestinal tumors, but are sensitive to MNU-induced intestinal tumorigenesis, PMS2+/ mice may be useful as a model to study environmental carcinogenic factors. This issue deserves further investigation using chronic sub-acute or low dose of carcinogen by oral route to mimic the human natural environmental carcinogen exposure.
One of the questions raised by our results was whether the PMS2+/ mice would show loss of the normal allele at the MNU exposure, as occurs in humans with the HNPCC syndrome, who inherit a single mutant allele of one of the mismatch repair genes and who develop a malignancy with loss of the remaining allele. To investigate the status of the remaining normal PMS2 allele in the tumors, microsatellite instability (MSI) analysis was performed in the 24 MNU-induced intestinal tumors in PMS2+/ mice and seven tumors in PMS2+/+ mice, using selective UV radiation fractionation (SURF) microdissection procedures and subsequent PCR as described (17). Briefly, relatively pure populations of 200400 cells (covered by a SURF dot) were selected from each intestinal tumor or from normal small intestine mucosa and the extracted DNA was diluted before PCR to determine the presence of novel sized microsatellite alleles. The microsatellite locus used for this assay was DXmit129, a CA-repeat locus on mouse chromosome X. No changes in repeat lengths were detected from the approximately 20 alleles measured from each tumor. Since many human colorectal adenomas as well as normal tissue from MSH2/ mice do not display microsatellite instability when analyzed with standard (non-dilution) detection techniques (12,1820), it is difficult to detect MSI except by dilution to single molecules prior to PCR. Prior analysis (17) demonstrated that altered allele lengths are readily detected at the locus, DXmit129, even in the normal mucosa of 7-week-old PMS2/ mice using the established SURF microdissection procedures. As a characteristic of MMR deficiency, MSI is detected in PMS2-deficient mouse tissue and both adenomas and normal mucosa from PMS2//Min mice, while stable microsatellite sequences were observed in both adenomas and surrounding normal mucosa from Min and PMS2+//Min mice (15). However, widespread microsatellite instability is probably not a major factor leading to accelerated aberrant crypt focus and adenoma development in MSH2//Min mice. Somatic APC mutations and loss of heterozygosity of APC are responsible for the additional intestinal tumors (14). Loss of heterozygosity of the Min allele was also observed in 25 of 29 (86%) of the intestinal tumor samples analyzed in PMS2+//Min mice (15). Loss of both copies of the APC gene, either by somatic mutations or by inactivation of the second allele, appears to be a prerequisite for intestinal tumor development in human and Min mice (2123).
Interestingly, in a different MSH2-deficient model, while MSH2+/ mice had an elevated extra-intestinal tumor incidence that rarely correlated with loss of the wild-type MSH2 allele, loss of the wild-type MSH2 allele was found in a significant fraction (818%) of the intestinal tumors in MSH2+//Min mice (16). The status of MMR in the intestinal tumors originating from MLH1+/ and MLH1+//APC1638N mutant mice was not investigated (13). In the present study, we found that MSI was negative in the intestinal tumors from MNU-treated PMS2+/ mice. Since our MSI assay is highly sensitive, our data suggest that mismatch repair was intact for the MNU-induced intestinal tumors, which developed between 48 and 53 weeks after MNU injection, and that the normal PMS2 allele was not inactivated by a single MNU treatment. In colon tumors of HNPCC patients, MSI was predominantly found in carcinomas rather than adenomas (24), indicating that in HNPCC, loss of MMR is a relatively late event occurring in adenomas and accelerating progression to carcinomas. PMS2+/ mice may be useful as a model to study the mechanisms that contribute to the development of MMR-deficient intestinal tumors in HNPCC patients.
The mechanisms of GI tumor development and lymphoid tumor development are clearly distinct in both humans and mice. Since PMS2/ mice do not develop spontaneous intestinal tumors, they do not appear to accumulate sufficient mismatches in intestine mucosa cell DNA to result in gene mutations or deletions in genes critical to intestinal carcinogenesis such as APC. However, carcinogen exposure may surpass this threshold to induce GI tumors. Because of the long latency required for induction and progression of GI tumors and the hypersensitivity of PMS2/ mice to MNU-induced lymphomas (25,26), PMS2/ mice are not suitable for MNU-induced GI carcinogenesis studies. However, in a separate study reported in abstract form (27), we have found that PMS2/ mice are hypersensitive to induction of intestinal tumors after exposure to another methylating agent, azoxymethane, a colon carcinogen.
Mutagenic mismatched base pairs may be produced by DNA polymerase misinsertion during DNA replication or may be induced following base modification by environmental mutagens. We propose the following mechanism for MNU-induced carcinogenesis in PMS2-deficient mice. MNU is an alkylating agent, producing various DNA adducts, among which O6-methylguanine (O6-meG) is the most important (2830). O6-meG in double-stranded DNA preferentially pairs with thymine (T) instead of cytosine (C) by any of the polymerases, producing O6-meG:T mispairs (31,32). During the subsequent round of repair or replication synthesis, this results in a G
A transition mutation. In MMR-proficient cells, O6-meG:T mispairs are recognized, resulting in single-strand patch formation opposite the O6-meG because this is in the newly synthesized strand. But, during repair synthesis, a polymerase incorporates a T opposite each O6-meG. This futile cycle is associated with DNA strand breaks whenever the single strand extends to replication site, either leading to induction of apoptosis and cell death or non-homologous recombination and chromosomal rearrangements at the site of the patches. For this reason, both G
A mutations and chromosomal rearrangements may contribute to the carcinogenesis of methylating agents in MMR-competent animals. As an additional factor present in the thymus, compensatory proliferation of the surviving cells due to cell loss from apoptosis may contribute to thymic lymphomagenesis.
In the setting of MMR deficiency, the exact transforming event that follows methylating agent induced DNA damage remains ill defined. Since MMR is not initiated at the site of MNU-induced O6-meG DNA lesions, cells survive with high levels of DNA methylation. As a consequence, G
A point mutations are formed in certain oncogenes such as K-ras or tumor suppressor genes such as APC and may be oncogenic. This tolerance to methylating agents is scored as drug resistance in cell lines and tumors (33,34), but our data indicate that the large number of G
A mutations results in a marked increase in tumorigenesis. Our results support the hypothesis that MMR-deficient cells are hypersensitive to the carcinogenic effects of alkylating agents.
In the setting of heterozygous expression of MSH2, lacI-bearing lambda shuttle phage transgenic (MSH2+//BC-1) mice were reported to have a similar lacI mutation frequency after MNU treatment, compared with those of control MSH2+/+/BC-1 mice in various tissues, whereas mice deficient in MSH2 (MSH2//BC-1 mice) were hypersensitive to mutation induction by this agent (35). These results suggested that HNPCC carriers may not be at increased risk of alkylation-induced mutations, and that spontaneously arising MMR-deficient cells would be predicted to accumulate mutations at a greatly accelerated rate on exposure to specific types of alkylating agents. In contrast, our present study demonstrates that methylating agents induce a high incidence of intestinal tumors in PMS2+/ mice compared with that observed in normal mice, while the remaining normal allele is still intact, and that the incidence of thymic lymphomas remains the same in PMS2+/ and PMS2+/+ mice. Thus, MMR status has a major impact on methylating sensitivity. These data indicate that methylation damage, either endogenous or environmental, is significantly more carcinogenic in the absence or insufficiency of MMR and that organ-specific factors also influence the capacity of MMR to protect against methylating agents.
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Acknowledgments
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We would like to thank Dr R.M.Liskay, Department of Molecular/Medical Genetics, Oregon Health Sciences University, Portland, Oregon, for providing PMS2 gene knockout mice. This work was supported in part by USPHS Grants P30CA43703, RO1CA63193, RO1ES06288, RO1CA73062 and UO1CA75525.
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Notes
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2 To whom correspondence should be addressed Email: slg5{at}po.cwru.edu 
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Received August 23, 1999;
revised December 9, 1999;
accepted December 20, 1999.