ARTICLE

1,2-Dimethylhydrazine-Induced Colon Carcinoma and Lymphoma in msh2-/- Mice

Claudia Colussi, Silvia Fiumicino, Alessandro Giuliani, Sandra Rosini, Piero Musiani, Caterina Macrí, Christopher S. Potten, Marco Crescenzi, Margherita Bignami

C. Colussi, S. Fiumicino, A. Giuliani, C. Macrí, M. Crescenzi, M. Bignami, Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanitá, Rome, Italy; S. Rosini, P. Musiani, Department of Oncology and Neurosciences, G. D'Annunzio University, Chieti, Italy; C. S. Potten, Paterson Institute for Cancer Research, Christie Hospital Trust, Manchester, U.K.

Correspondence to: Margherita Bignami, Ph.D., Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanitá, Viale Regina Elena 299, 00161 Rome, Italy (e-mail: bignami{at}iss.it).


    ABSTRACT
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Defective mismatch repair (MMR) in humans is particularly associated with familial colorectal cancer, but defective repair in mice is generally associated with lymphoma in the absence of experimental exposure to carcinogens. Loss of MMR also confers resistance to the toxic effects of methylating agents. We investigated whether resistance to methylation contributes to increased susceptibility to colorectal cancer in mice by exposing mice with defects in the MMR gene msh2 to a methylating agent. Methods: Tumor incidence and time of death in msh2+/+, msh2+/-, and msh2-/- mice were analyzed after weekly exposure (until tumor appearance) to the methylating agent 1,2-dimethylhydrazine (DMH). Chemically induced and spontaneous tumors were characterized by frequency, type, and location. The tumor incidence in untreated and treated mice of each genotype was compared by a Mann–Whitney U test. Carcinogen-induced apoptosis in histologic sections of small and large intestines was also determined. All statistical tests were two-sided. Results: Homozygous inactivation of the msh2 gene statistically significantly accelerated (P<.0001) death due to the development of DMH-induced colorectal tumors and lymphomas. Rates of death from DMH-induced colorectal adenocarcinoma were similar in msh2 heterozygous and wild-type mice, but only msh2 heterozygotes (msh+/-) developed additional, noncolorectal malignancies (notably trichofolliculoma [two of 21], angiosarcoma of the kidney capsule [two of 21], and lymphoma [one of 21]), suggesting that heterozygosity for msh2 slightly increases DMH susceptibility. DMH induced apoptosis in small intestinal and colonic epithelial crypts that was dependent on active msh2. Conclusions: Inactivation of msh2 allows the proliferation of gastrointestinal tract cells damaged by methylating agents. Furthermore, MMR constitutes a powerful defense against colorectal cancer induced by DNA methylation.



    INTRODUCTION
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mismatch repair (MMR) system carries out surveillance and repair of DNA replication errors (1). The MSH2, MSH6, MLH1, and PMS2 genes are all required for MMR. An inherited mutation in one allele of an MMR gene is associated with a predisposition for hereditary nonpolyposis colorectal cancer (HNPCC) syndrome (2). Individuals with HNPCC develop colon, endometrial, and other cancers in which the remaining active allele of the altered gene is inactivated and HNPCC tumors are devoid of MMR capacity [for a review, see (3)]. MMR-defective tumor cells have a mutator phenotype due to the persistence of numerous replication errors in both coding and noncoding (microsatellite) DNA sequences. The mutator phenotype is thought to favor cancer development by increasing the probability of mutations in tumor suppressor genes and/or genes controlling proliferation or apoptosis. MMR-defective mice die, at early age, mainly of lymphoid tumors. The increased cancer susceptibility of mice defective in msh2, mlh1, msh6, or pms2 is consistent with a causative relationship between a mutator phenotype and carcinogenesis (4–7).

Loss of MMR also confers resistance to the toxic effects of methylating agents (8,9). These compounds are carcinogenic, mutagenic, and cytotoxic because they methylate guanine bases in DNA to produce O6-methylguanine (O6-MeG). O6-MeG can be removed from DNA by a specific repair enzyme, the O6-methylguanine DNA methyltransferase (MGMT) (10), although MGMT levels are low in some human tissues (11). O6-MeG in DNA may be recognized by MMR (12,13), and this interaction activates apoptotic cell death (14–16). MMR-defective human or mouse cells are, consequently, resistant (tolerant) to the cytotoxic effects of methylating agents, and O6-MeG can accumulate in the DNA of MSH2-, MSH6-, MLH1-, or PMS2-defective cells without lethal effects [for a review, see (17)]. Persistent DNA O6-MeG produces G-to-A transitions (18). Thus, MMR normally plays an important role in the elimination of potentially mutated cells (19).

It is not presently known whether the loss of O6-MeG-related apoptosis, which accompanies tolerance to methylation damage, plays a role in the increased cancer risk associated with defective MMR. Many methylating agents are powerful carcinogens that generate O6-MeG in DNA. O6-MeG has been found in DNA of normal human colon (20), suggesting that colon cells might be at risk of persistent DNA methylation. An association between low MGMT levels and colon cancer risk has even been postulated (20–22).

We have investigated a possible contribution of DNA methylation damage to the development of colorectal cancer in MMR-defective mice. As an experimental model, we used mice that were null (msh-/-), heterozygous (msh+/-), or wild-type (msh+/+) for the MMR gene msh2. Tumor induction by repeated exposures to the methylating carcinogen 1,2-dimethylhydrazine (DMH) was compared in the three genotypes. DMH is a procarcinogen that, after metabolic activation, induces formation of O6-MeG in the DNA of several tissues, including colon, ileum, and liver (23), and induces tumors in the colorectum (24,25).

The tumors that arise spontaneously in msh2-/- knockout mice are overwhelmingly lymphomas (5,26,27). The few surviving animals develop adenocarcinomas of the duodenum and jejunum and skin neoplasms (27,28). We hypothesized that the increased resistance to methylating agent-induced apoptosis is a factor in tumor susceptibility of MMR-defective mice. We examined the effect of DMH, an acknowledged colon carcinogen, in msh2-deficient animals. The incidence of both colorectal tumors and lymphoma and the time to death were analyzed, and the apoptotic response in the proliferating cells of the colonic epithelium was examined.


    MATERIALS AND METHODS
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mice.

MMR-deficient mice (provided by Professor H. te Riele, The Netherlands Cancer Institute, Amsterdam) carrying a targeted disruption in exon 12 of the msh2 gene were described previously (5). 129OLA/FVB msh2-/- mice were crossed five times with FVB mice to obtain an almost 95% FVB genetic background. All of the animals were fed a standard laboratory diet and water ad libitum and were maintained on a 12-hour light–12-hour dark schedule. The animal experimental protocol has been reviewed and approved by the Italian Ministry of Health.

Genotyping of mice by polymerase chain reaction (PCR).

Pieces of tail were digested overnight at 53 °C in 700 µL of lysis buffer (i.e., 50 mM Tris [pH 8], 100 mM EDTA, 100 mM NaCl, 1% sodium dodecyl sulfate, and 1 mg/mL proteinase K). DNA was extracted by standard procedures and amplified in a three-primer PCR reaction (50 µL) containing 125 pmol of each primer, 200 µM deoxynucleoside triphosphates, 2 mM MgCl2, and 1.25 U of Taq polymerase. Primers were as described previously (28). The reaction conditions were 94 °C for 5 minutes, 60 °C for 2 minutes, and 72 °C for 2 minutes (for one cycle); 94 °C for 1 minute, 60 °C for 2 minutes, and 72 °C for 2 minutes (for 40 cycles); and 94 °C for 1 minute, 60 °C for 2 minutes, and 72 °C for 10 minutes (for one cycle). The wild-type and targeted msh2 alleles generate a 164- and a 194-base-pair PCR product, respectively.

Induction of tumors.

The carcinogenic treatment was performed as described previously (24). Briefly, DMH (Sigma Chemical Co., St. Louis, MO) was dissolved (2 mg/mL) immediately before use in 1 mM EDTA (pH 7). Groups of mice, 4–5 weeks of age (12 msh2+/+, 12 msh2-/-, and 21 msh2+/- animals), received weekly subcutaneous injections of DMH (20 mg/kg body weight) until tumors appeared. Control animals (12 msh2+/+, 12 msh2-/-, and 15 msh2+/-) received mock injections of 1 mM EDTA.

Pathologic analysis.

Complete autopsy was performed on all mice that were found dead or that were killed by cervical dislocation when they were moribund. Before being embedded in paraffin blocks, tumors and tissues were removed and fixed in 10% formaldehyde in phosphate-buffered saline. The sections were stained with hematoxylin–eosin and examined.

Statistical analysis.

A two-sided Mann–Whitney U test on the rank sums was used to compare intervals from the beginning of treatment to the time of death between treated and untreated mice (29). This test was adopted to avoid the imposition of any distributional hypothesis on the data. The probability of survival was also estimated by the standard Kaplan–Meier parametric approach, and the difference in median time to death between groups was analyzed with two-sided Wilcoxon and log-rank tests. Computation was performed by the NPAR1WAY procedure of the SAS system 8.00 version for personal computers (Statistical Analysis Software, Inc., Cary, NC). All statistical tests were considered to be statistically significant at P<.05.

Immunohistochemistry.

Paraffin-embedded intestinal sections (5 µm) were baked at 65 °C for 1 hour, deparaffinized, and rehydrated through a series of graded alcohols. Sections were incubated overnight at 4 °C with the msh2 antiserum (a gift from Professor J. Jiricny, University of Zurich, Zurich, Switzerland) in 0.1 M sodium phosphate (pH 8) with 0.5% Nonidet P-40 and 5% nonfat dry milk. The sections were then incubated with fluorescein-conjugated, goat anti-rabbit immunoglobulin antiserum and counterstained with Hoechst 33258.

Apoptosis scoring.

Animals (10–12 weeks of age) were killed 8 or 16 hours after injection with DMH (80 mg/kg). Small and large intestines were removed, flushed with water, fixed in Carnoy's fixative for 30 minutes, and stored in 70% ethanol. Apoptosis was scored in histologic sections as described previously (30). Briefly, 25 good longitudinal sections were selected per animal and divided down the long axis into two half crypts, which were scored separately; 50 such half-crypt sections per mouse were scored. Starting at the base of the crypt column, the cells were numbered up each side, and data were recorded on a cell-position-by-cell-position basis. The location of each apoptotic event was then recorded in term of its position. The results are presented as plots of frequency of apoptotic events at each position in the crypt. Each frequency plot consists of the pooled data from three mice.

Microsatellite analysis.

The tumor was microdissected from formalin-fixed, paraffin-embedded specimens, and DNA was extracted by incubating individual 5-µm histologic sections in 0.5 mL of buffer containing 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, bovine serum albumin (10 mg/mL), 1% Tween 20, and proteinase K (10 µg/mL). The sections were incubated overnight at 55 °C, boiled for 5 minutes, and then cooled on ice. Normal tissue DNA was extracted from the tails. DNA aliquots were amplified with end-labeled primers and analyzed by electrophoresis on denaturating 6% polyacrylamide gels.


    RESULTS
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
DMH-Induced Tumorigenesis in msh2-Deficient Mice

Four- to five-week old, wild-type, msh2+/- or homozygous msh2-/- knockout mice were treated weekly with DMH. The times at which tumor-bearing animals died or were killed are summarized in Fig. 1Go, A. DMH-treated msh2-/- animals developed tumors, with a median time to death of 83.5 days (95% confidence interval [CI] = 62 to 112 days) from the start of the treatment. This latent period was statistically significantly reduced (P<.0002, Mann–Whitney U test) compared with untreated knockout animals, all of which developed lymphoblastic lymphoma after a median time of 151.5 days (95% CI = 131 to 181 days) (P<.001, log-rank and Wilcoxon tests). Pathologic analysis indicated a clear difference in the tumor load of DMH-treated and untreated knockout animals. Although all knockout animals developed lymphomas, colorectal adenocarcinomas were present in six of 12 DMH-treated msh2-/-mice. The adenocarcinomas, which were located predominantly in the lower part of the colon or in the rectum, were not observed in untreated knockout animals (none of 12). No difference in tumor distribution was seen between the sexes (carcinomas were in three males and three females). The lower part of the colon and the rectum showed multiple papillary or sessile adenomas with areas of dysplasia and carcinoma in situ close to overt, usually exophytic, adenocarcinomas. Multiple areas of atypia were found, suggesting that several initiating events were induced by the carcinogen. In a few cases, multiple separate tumors were observed. Histologically, the colorectal carcinomas were moderately to well differentiated, and their growth was frequently limited to the mucosa (Fig. 2Go), although submucosal and lymphatic invasion was evident in some mice.



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Fig. 1. Survival curves of 1,2-dimethylhydrazine (DMH)-treated animals. Comparisons were done with the use of the Mann–Whitney U, log-rank, and Wilcoxon tests. A) Data from 12 untreated msh2-/- and 12 DMH-treated msh2-/- mice are plotted. The median time to death was 151.5 days (95% confidence interval [CI] = 131 to 181 days) for untreated msh2-/- mice and 83.5 days (95% CI = 62 to 112 days) for DMH-treated msh2-/- mice. At those times, the number of animals at risk was six in both groups. B) Groups of 12 msh2+/+ and 21 msh2+/- mice were treated with DMH as described in the "Materials and Methods" section and killed when moribund. The median time to death was 209 days (95% CI = 196 to 217 days) and 204 days (95% CI = 192 to 210 days) for msh2+/+ and msh2+/- mice, respectively. At those times, the number of animals at risk was six and 10 in msh2+/+ and msh2+/- mice, respectively. P values, calculated according to the indicated methods, are shown. Error bars show 95% CIs at 25% and 50% survival.

 


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Fig. 2. Colon adenocarcinoma. Tumor tissue showing crypt structure disorganization. The colon histology of untreated msh2-/- mice (A) shows a normal flat mucosa punctuated by numerous straight tubular crypts that extend down to the muscularis mucosa. The crypts contain abundant goblet cells. The histology of the colonic mucosa of 1,2-dimethylhydrazine-treated mice (B) shows many neoplastic crypts and glands irregular in shape and size with a tendency to an exophytic growth. Crypts are lined by a tall, hyperchromatic, somewhat disordered epithelium without differentiation into mature goblet cells. Hematoxylin–eosin. Original magnifications x400 (panel A) and x200 (panel B).

 
Thus, exposure to DMH accelerated death from the lymphomas to which msh2-/- mice normally succumb. In addition, the selective carcinogenicity of DMH toward the colon is apparent in msh2-/- mice.

In contrast to the msh2-/- mice, neither heterozygous nor wild-type mice were prone to lymphoma development. DMH-treated msh2+/- mice died at a median time to death of 204 days (Fig. 1Go, B). All (21 of 21) heterozygous mice had colon adenocarcinomas at death; a lymphoma was present in a single case. There were two examples of lung metastasis and two of angiosarcomas of the kidney capsule, which, although uncommon, have been reported previously in mice exposed to DMH (31) (Table 1Go). Two mice also developed trichofolliculomas resembling the sebaceous tumors that form a subset of spontaneous tumors in msh2-/- animals (26). These trichofolliculomas are reminiscent of the skin malignancies associated with the Muir–Torre variant of HNPCC (2).


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Table 1. Tumor distribution in untreated or 1,2-dimethylhydrazine (DMH)-treated msh2-/-, msh2+/-, and msh2+/+ mice*
 
As expected, all DMH-treated msh2+/+ mice (12 of 12) developed adenocarcinomas of the colon. Similar to a previous report (24), the median time to death was 209 days (95% CI = 196 to 217 days) from the beginning of treatment. This value did not differ statistically significantly from the 204 days (95% CI = 192 to 210 days) observed for heterozygous animals (P = .39, Mann–Whitney U test). No cases of lymphomas were noted in this group (Fig. 1Go, B; Table 1Go). There was no difference among the mice with the three genotypes in the distribution of colorectal neoplasms, all of which were situated in the distal part of the large bowel. The colorectal tumors of wild-type and heterozygous animals were larger than those arising in msh2-/- mice, suggesting that the latter animals died of lymphoma.

The absence of msh2, therefore, increases the tumorigenicity of DMH in mice. This absence affects the colon, and DMH-induced colorectal adenocarcinomas developed in msh2-/- animals in about one third of the time required in wild-type animals. A statistically significant difference in the times to death (P<.0001, log-rank, Wilcoxon, and Mann–Whitney U tests) was observed between DMH-treated msh2-/- and msh2+/+ mice with a median time to death of 83.5 days (95% CI = 62 to 112 days) and 209 days (95% CI = 196 to 217 days), respectively. The lymphomas to which msh2-/- mice are already predisposed also exhibited an earlier onset following DMH treatment. The accelerated induction of colorectal tumors and lymphomas by DMH is dependent on the inactivation of both msh2 alleles and was not observed in msh2+/- mice. The somewhat broader spectrum of tumors in the DMH-treated heterozygous animals is consistent with a modest general increase in carcinogen susceptibility as a consequence of the retention of a single active msh2 allele.

The similarity in response of the msh2+/- and wild-type mice suggests that heterozygosity for msh2 is not an important factor in DMH-induced carcinogenesis. In agreement with this suggestion, we found no evidence for microsatellite instability in DMH-induced tumors from heterozygous animals. Four colorectal adenocarcinomas from DMH-treated msh2+/- mice were analyzed at the D17mit123 locus, which is closely linked to msh2. The D18mit14 locus was also examined in two of the four tumors. Neither locus was mutated in any of the tumors. Four spontaneous lymphomas from msh2-/- mice displayed the expected microsatellite instability. D17mit123 and at least one other microsatellite locus were mutated in each case (Fig. 3Go).



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Fig. 3. Microsatellite instability analysis of normal and tumor tissues from msh2+/+, msh2+/-, and msh2-/- mice at the indicated loci. N = normal tissue; T = tumor tissue. Arrows indicate the samples with alterations in microsatellite length.

 
Although derived from limited numbers, these data suggest that the DMH-induced colorectal adenocarcinomas do not display the microsatellite instability that is associated with msh2-/- tumors. Lack of microsatellite instability suggests that the active msh2 allele is retained in DMH-induced colorectal adenocarcinomas of msh2+/- mice.

Msh2 Expression and Apoptosis in the Gastrointestinal Tract of Wild-Type and msh2 Knockout Animals

Msh2 protein was detected in the lower two thirds of immunohistochemically stained colonic crypts, from position 1 to approximately position 30, from both msh2+/+ and msh2+/- animals (Fig. 4Go, A and B). msh2 was detected uniformly in the nucleus; the intensity or distribution of msh2 protein in heterozygous and wild-type mice was similar (Fig. 4Go, A and B). As expected, no staining was detected in crypts from homozygous knockout animals (Fig. 4Go, C).



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Fig. 4. Immunohistochemistry of the colon in msh2+/+ (A), msh2+/- (B), and msh2-/- (C) mice; msh2 staining is apparent in the lower two thirds of the colonic crypts. Original magnification x400.

 
DMH induced apoptosis in the colon and in the small intestine (Fig. 5Go, A–G). The extent of apoptosis varied with the time after carcinogen exposure, and the spatial distribution of apoptotic cells was asymmetric. At early times after DMH administration (8 hours), the apoptotic index in the large intestine was around 15% and was maximal at crypt positions 4 and 5, which is the proliferative, non-stem-cell compartment in this tissue (Fig. 5Go, A–C and E). The apoptotic index declined to background levels by position 10. Early DMH-induced apoptosis (8 hours) was essentially independent of msh2 expression, and the levels were similar in crypts derived from msh2+/+, msh2+/-, and msh2-/- mice. By 16 hours, the apoptotic index had increased to approximately 50%, was again maximal at positions 4 and 5, and declined to background levels at around position 10. DMH-induced cell death at 16 hours was mostly dependent on msh2, and an apoptotic index of approximately 10% was observed in homozygous knockout animals at 16 hours time (Fig. 5Go, F).



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Fig. 5. Frequency of apoptosis in the crypts of the large and small intestines after 1,2-dimethylhydrazine (DMH) exposure. Cohorts of msh2+/+ and msh2-/- animals were given subcutaneous injections of DMH (80 mg/kg). Schematics of the structures of colonic (A) and small intestinal (D) crypts are shown. Cells in the crypt are given a number starting with number 1 at the crypt base. Each point represents the mean and standard deviation from 50 cells from each of three mice. The 95% confidence intervals (CIs) of the mean apoptotic indices at positions 4 and 5 of the intestinal crypts, where most of the apoptosis is located, are indicated. Panel A: large intestine—the slowly dividing stem (S) cells, which are located at the very base of the crypt (positions 1 and 2), divide to give the rapidly proliferating transit (T) cells, which further divide to produce the mature cells of the crypt. Panel B: normal colonic crypts. Original magnification x400. Panel C: apoptotic cells in the crypt section of the large intestine stained with hematoxylin–eosin. Original magnification x400. Panel D: small intestine—the base consists of differentiated Paneth cells; above these cells are the S cells (positions 4 and 5) and then the T cells (positions 5 to about 15–20); above position 20, cells differentiate and migrate along the crypt and into the villus. Panel E: apoptosis of the colon 8 hours after DMH treatment. The mean apoptotic indices at positions 4 and 5 for msh2+/+ mice are 12 (95% CI = 8.6 to 15.4) and 12 (95% CI = 6.3 to 17.6), respectively; the corresponding values for msh2-/- mice are 17 (95% CI = 15.9 to 18.1) and 10 (95% CI = 5.5 to 14.5). Panel F: apoptosis of the colon 16 hours after DMH treatment. The mean apoptotic indices at positions 4 and 5 for msh2+/+ mice are 52 (95% CI = 36.2 to 67.8) and 23 (95% CI = 21.7 to 24.2), respectively; the corresponding values for msh2-/- mice are 10 (95% CI = 8.7 to 11.3) and 12 (95% CI = 10.7 to 13.3). Panel G: apoptosis of the small intestine 8 hours after DMH treatment. The mean apoptotic indices at positions 4 and 5 for msh2+/+ mice are 25 (95% CI = 19.3 to 30.6) and 16 (95% CI = 14.7 to 17.3), respectively; the corresponding values for msh2-/- mice are 10 (95% CI = 5.5 to 14.5) and 8 (95% CI = 6.7 to 9.3).

 
DMH also induced apoptosis in the crypts of the small intestine. Similar apoptotic indices were found in wild-type and heterozygous animals (data not shown). In contrast to the colon, even 8 hours after treatment, most of the DMH-induced apoptosis observed in the small intestine was msh2 dependent, and very few apoptotic figures were observed in msh2-/- animals (Fig. 5Go, G). Apoptosis was predominantly confined to the stem cell compartment, positions 4 and 5 (Fig. 5Go, D).

Thus, with the exception of a wave in colonic crypt cells at early times after treatment, DMH-induced apoptosis in the small or large intestine was dependent on functional msh2 protein.


    DISCUSSION
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although previous studies (27,32,33) showed an increased overall cancer susceptibility of msh2-/-, mlh1-/-, or pms2-/- mice to alkylating agents, the data presented here provide, to our knowledge, the first example of a decreased time to death from colon cancer in carcinogen-treated MMR-deficient mice. Homozygous inactivation of msh2 increases the susceptibility of mice to the organ-specific methylating carcinogen DMH. DMH-induced apoptosis in the colon, the target organ for this carcinogen, is largely controlled by msh2 protein. DMH induces different patterns of apoptosis in crypt cells of target (colon) and nontarget (small intestine) organs, which has been proposed as a possible factor in the organ specificity of DMH carcinogenesis (34). Resistance to DMH-induced apoptosis is associated with an increased susceptibility to DMH-induced colon cancer in msh2 knockout mice.

The organ specificity of DMH is not, however, related in a simple way to apoptosis. Although an early wave of apoptosis in colonic crypt cells was largely independent of msh2, most DMH-induced cell death in both colon and small intestine was msh2 dependent. This finding is consistent with the report of Toft et al. (28), which indicated that methylating agent-induced apoptosis in mouse small intestinal crypt cells is dependent on functional msh2. Despite this association, we did not observe DMH-induced tumors of the small intestine. It appears likely that msh2, by controlling apoptosis, provides a general protection mechanism against DNA methylation damage in all mouse tissues. Additional factors, such as MGMT level and turnover rate or the renewal rate of the cell population affected by DMH, may underlie the colon specificity of this carcinogen.

Differences in apoptosis clearly do not explain the organotropism of DMH carcinogenesis. The overwhelming tendency of DMH to induce colorectal tumors is apparently independent of the msh2 background. We did observe, however, accelerated colorectal carcinogenesis in msh2 knockout animals. This finding may reflect the decreased susceptibility to O6-MeG-induced apoptosis that accompanies loss of msh2. Thus, increased resistance to methylation-induced apoptosis (14–16,35) and increased spontaneous and DNA damage-related mutation rates (19) might contribute to the development of colorectal tumors. It is important also to stress that inactivation of msh2 dramatically enlarges the number of colonic crypts cells that are target for neoplastic transformation by DMH.

The pattern of tumors in the homozygous msh2-/- mice reflected the expected colorectal specificity of DMH and the spontaneous susceptibility of msh2-/- animals to lymphoma. In the msh2+/- animals, however, the pattern was more complex. Although the majority of DMH-treated heterozygous mice died of colorectal carcinoma, a substantial minority developed tumors at additional sites. Angiosarcoma of the kidney capsule in two of 21 msh2+/- mice suggests that heterozygosity for msh2 might increase the susceptibility to DMH carcinogenesis in tissues other than the colon. Angiosarcoma of the kidney capsule is associated, although rarely, with DMH exposure (31,36). Furthermore, the skin neoplasms and the lymphomas are reminiscent of spontaneous tumors associated with complete loss of Msh2 function (26). A similar heightened susceptibility to induction of nonhematologic malignancies by N-ethyl-N-nitrosourea, a primarily lymphomagenic carcinogen, might underlie the more heterogeneous pattern of tumors in msh2+/- animals (27). Thus, although a single active msh2 allele is compatible with a normal life span and is sufficient to provide protection against endogenous carcinogens, it may be insufficient to protect against high doses of a potent carcinogen. MGMT levels, which differ widely among organs, might be the crucial factor in determining tumor susceptibility (32).

In agreement with previous studies (27,37), we found no evidence of early inactivation of the active MMR allele in the tumors that developed in heterozygous mice. This finding contrasts dramatically with results seen with HNPCC, in which inactivation of the functional allele is considered to be a probable early event in tumor development. Retention of an active msh2 allele suggests that selection for resistance to methylation damage—the starting point for this study—might not underlie the emergence of tumors in heterozygous individuals with HNPCC. There are two obvious caveats, however. First, the mice were exposed repeatedly to high doses of a colon-specific carcinogen. This dosing contrasts with the chronic, low-level exposure to DNA-methylating agents that human colon cells might encounter and that could provide a selective pressure for MMR loss. In addition, mouse cells are generally less sensitive to killing by methylating agents than are their human counterparts, and loss of MMR confers only a modest (twofold) increased resistance (38). This small increase compares with the greater than 50-fold higher resistance of MMR-defective human cells. The selective advantage provided by inactivation of the second MMR allele is, therefore, minor in mouse cells, and other targets of mutational inactivation may contribute more substantially to tumor development.

DMH also markedly decreases the latent period for lymphoma development in msh2-/- mice. This finding suggests that MMR provides substantial protection against lymphomagenesis by alkylating carcinogens (27,32). We note that the constitutional absence of MMR predisposes humans to hematologic malignancy (non-Hodgkin's lymphomas and acute myeloid leukemia) and type I neurofibromatosis (39,40). Thus, although the parallels between heterozygous msh2 mice and HNPCC are not strong, homozygous knockout mice are good models for their human counterparts.

DNA methylation damage has long been suspected to be a contributor to human colon cancer. We conclude that MMR constitutes a powerful defense against colorectal cancer induced by an exogenous methylating carcinogen. Our data support the notion that failure by MMR to elicit the apoptotic pathway associated with persistence of this methylated base is associated with an increased risk of colon cancer.


    NOTES
 
This work is dedicated to the memory of Franco Tatò who transmitted and shared the enthusiasm for discoveries and will be missed immensely by all of us.

Supported by a grant from Associazione Nazionale Ricerca sul Cancro and Ministero della Sanitá. C. Colussi was partially supported by a fellowship from Federazione Italiana Ricerca sul Cancro.

We are grateful to P. Karran (Imperial Cancer Research Fund, U.K.) for helpful discussions and Professor Mariani Costantini (University of Rome "La Sapienza," Italy) for critical review of the manuscript. M. Bignami thanks her mentors, Umberto Saffiotti and Ruggero Montesano, for guidance and support early in her career.


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received January 29, 2001; revised July 2, 2001; accepted August 16, 2001.


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