Candidate mutator genes in mismatch repair-deficient thymic lymphomas: no evidence of mutations in the DNA polymerase delta gene

DNA mismatch repair (MMR) proteins recognize nucleotides that are incorrectly paired. Deficiencies in MMR lead to increased genomic instability reflected in an increased mutation frequency and predisposition to tumorigenesis. Mice lacking the MMR gene, Msh2, develop thymic lymphomas that exhibit much higher mutational frequencies than other Msh2(-/-) tumours and Msh2(-/-) normal thymic tissue, suggesting that an additional mutator may have been acquired in a tissue-specific manner. Clustered mutations observed exclusively in the thymic lymphomas suggests that a gene(s) associated with the replication machinery might have become altered during tumorigenesis. Based on mutation studies in Saccharomyces cerevisiae lacking Msh2 and DNA polymerase delta (DNA pol delta), we hypothesized that the acquisition of mutations in DNA pol delta could contribute to the hypermutator phenotype and tumorigenesis in Msh2(-/-) thymic tissue. Furthermore, previous reports have suggested that genes containing mononucleotide repeats are non-random mutational targets in the absence of MMR. Therefore, we sequenced all 26 exons of the DNA pol delta catalytic subunit, including the six exons containing mononucleotide repeats of >5 bp, from nine Msh2(-/-) thymic lymphomas and two wild-type controls. No DNA pol delta pathogenic mutations were found in the thymic lymphomas, although several DNA base differences compared with published DNA pol delta sequences were observed. We conclude, therefore, that inactivating mutations in DNA pol delta are not a contributing factor in the development of the hypermutator phenotype in MMR-deficient murine thymic lymphomas.     

Mutagenesis in PMS2- and MSH2-deficient mice indicates differential protection from transversions and frameshifts

DNA mismatch repair (MMR) deficiency leads to an increased mutation frequency and a predisposition to neoplasia. 'Knockout' mice deficient in the MMR proteins Msh2 and Pms2 crossed with mutation detection reporter (supF, lacI and cII) transgenic mice have been used to facilitate a comparison of the changes in mutation frequency and spectra. We find that the mutation frequency was consistently higher in Msh2-deficient mice than Pms2-deficient mice. The lacI target gene, which is highly sensitive to point mutations, demonstrated that both Msh2- and Pms2-deficient mice accumulate transition mutations as the predominant mutation. However, when compared with Msh2(-/-) mice, lacI and cII mutants from Pms2-deficient mice revealed an increased proportion of +/-1 bp frameshift mutations and a corresponding decrease in transversion mutations. The supF target gene, which is sensitive to frameshift mutations, and the cII target gene revealed a strong tendency for -1 bp deletions over +1 bp insertions in Msh2(-/-) compared with Pms2(-/-) mice. These data indicate that Msh2 and Pms2 deficiency have subtle but differing effects on mutation avoidance which may contribute to the differences in tumor spectra observed in the two 'knockout' mouse models. These variances in mutation accumulation may also play a role, in part, in the differences seen in prevalence of MSH2 and PMS2 germline mutations in hereditary non-polyposis colorectal cancer patients.     

Msh2 DNA mismatch repair gene deficiency and the food-borne mutagen 2-amino-1-methy1-6-phenolimidazo [4,5-b] pyridine (PhIP) synergistically affect mutagenesis in mouse colon

Msh2 deficiency and food-borne carcinogen PhIP have been implicated as genetic and environmental factors, respectively, in human colon carcinogenesis. It is not clear whether loss of one or both alleles of Msh2 gene increases the mutational sensitivity in colon when exposed to environmental carcinogens. In the current study, Msh2(+/-)/lacI and Msh2(-/-)/lacI double transgenic mice were treated with PhIP and mutations in the lacI gene were studied in the colon. The spontaneous mutation frequency (MF) is approximately eightfold higher in Msh2(-/-) mice than in Msh2(+/+) mice, while Msh2(+/-) mice display similar levels of spontaneous mutation as the Msh2 wild type mice. PhIP induced a significant increase in MF in all genotypes of mice. However, induced MF is much higher in Msh2(-/-) mice compared to Msh2(+/+) and Msh2(+/-) mice. Msh2(+/-) mice displayed an increased level of G:C>T:A transversions and -1 frameshifts upon PhIP treatment. In contrast, loss of both Msh2 alleles mainly results in increased frequency of G:C>A:T transitions when exposed to PhIP. These results suggest that a defect in mismatch repair may result in an enhanced sensitivity from exposure to a dietary carcinogen. It also provides insight into interaction between genetic and environmental factors in human carcinogenesis.     

Elevated mutant frequencies and increased C : G-->T : A transitions in Mlh1-/- versus Pms2-/- murine small intestinal epithelial cells

Mutations in DNA mismatch repair (MMR) genes are associated with increased genomic instability and susceptibility to cancer. Mice rendered deficient in either Mlh1 or Pms2 as a result of gene targeting are prone to tumorigenesis, particularly, lymphomas. In addition, although Mlh1-/- mice also develop small intestinal adenomas and adenocarcinomas, Pms2-/- animals remain free of such tumors. To establish whether this phenotypic dichotomy might be associated with a quantitative and/or qualitative difference in genomic instability in these mice, we determined small intestinal epithelial cell DNA mutant frequency and mutation spectrum using a transgenic lambda-phage lacI reporter system. Mutant frequencies obtained from both Mlh1-/- and Pms2-/- mice revealed elevations of 18- and 13-fold, respectively, as compared to their wild-type littermates. Interestingly, we found that C : G-->T : A transitions were significantly elevated in Mlh1-/- mice, accounting in large measure for the 1.5-fold lacI mutant frequency increase seen in these animals. We hypothesize that the increased level of C : G-->T : A mutations may explain, in part, why Mlh1-/- mice, but not Pms2-/- mice, develop small intestinal tumors. Furthermore, the difference in the lacI mutational spectrum of Mlh1-/- and Pms2-/- mice suggests that other MutL-like heterodimers may play important roles in the repair of G : T mispairs arising within murine small intestinal epithelial cells.     

Elevated mutant frequencies and predominance of G:C to A:T transition mutations in Msh6(-/-) small intestinal epithelium

The DNA mismatch repair (MMR) system is primarily responsible for purging newly synthesized DNA of errors incurred during semi-conservative replication. Lesion recognition is initially carried out by one of two heterodimeric protein complexes, MutS(alpha) or MutS(beta). While the former, comprised of MSH2 and MSH6, recognizes mispairs as well as short (1-2 nucleotide) insertions/deletions (IDLs), the latter, made up of MSH2 and MSH3, is primarily responsible for recognizing 2-6 nucleotide IDLs. As most of the functional information on these heterodimers is derived from in vitro studies, it was of interest to study the in vivo consequences of a lack of MutS(alpha). To this end, Big Blue( trade mark ) mice, that carry a lacI(+) transgenic lambda shuttle-phage mutational reporter, were crossed with Msh6(-/-) mice to evaluate the specific contribution of MutS(alpha) to genome integrity. Consistent with the importance of MutS(alpha) in lesion surveillance, small intestine epithelial cell DNA derived from lacI(+) Msh6(-/-) mice exhibited striking increases (average of 41-fold) in spontaneous mutant frequencies. Furthermore, the lacI gene mutation spectrum was dominated by G:C to A:T transitions, highlighting the critical importance of the MutS(alpha) complex in suppressing this frequently observed type of spontaneous mutation.     

Differing patterns of genetic instability in mice deficient in the mismatch repair genes Pms2, Mlh1, Msh2, Msh3 and Msh6

Defects in genes associated with DNA mismatch repair (MMR) have been linked to hereditary colon cancer. Because the MMR pathway includes multiple factors with both overlapping and divergent functions, we sought to compare the impact of deficiencies in each of several MMR genes on genetic instability using a collection of knock-out mouse models. We investigated mutation frequencies and patterns in MMR-deficient mice using two transgenic reporter genes, supFG1 and cII, in the context of mice deficient for Pms2, Mlh1, Msh2, Msh3 or Msh6 or both Msh2 and Msh3 or both Msh3 and Msh6. We found that the mean mutation frequencies of all of the MMR-deficient mice were significantly higher than the mean mutation frequencies of wild-type mice. Mlh1-deficient mice and Msh2-deficient mice had the highest mutation frequencies in a comparison of the single nullizygous mice. Of all the mice studied, mice nullizygous for both Msh2 and Msh3 and those nullizygous for both Msh3 and Msh6 displayed the greatest overall increases in mutation frequencies compared with wild-type mice. Sequence analysis of the mutated reporter genes revealed significant differences between the individual groups of MMR-deficient mice. Taken together, our results further characterize the functions of the MMR factors in mutation avoidance and provide in vivo correlation to biochemical models of the MMR pathway.     

A lack of DNA mismatch repair on an athymic murine background predisposes to hematologic malignancy

Inheritance of a germline mutation in one of the DNA mismatch repair genes predisposes human individuals to hereditary nonpolyposis colorectal cancer, characterized by development of tumors predominantly in the colon, endometrium, and gastrointestinal tract. Mice heterozygous for a mismatch repair-null mutation generally do not have an increased risk of neoplasia. However, mice constitutively lacking mismatch repair are prone to tumor development from an early age, particularly thymic lymphomas. Mismatch repair-deficient mice crossed to Apc(+/-) mice develop an increased spontaneous intestinal tumor incidence, demonstrating that the tumor spectrum can be genetically influenced. Here, we bred Msh2- and Msh6-deficient mice to athymic nude mice, hypothesizing that a broader tumor spectrum may be observed if mice are able to survive longer without succumbing to thymic lymphomas. However, Msh2(-/-);Foxn1(nu/nu) and Msh6(-/-);Foxn1(nu/nu) mice developed primarily early-onset lymphoblastic lymphomas. Using B-cell-specific markers, we found these tumors to be predominately B-cell in origin. The development of hematologic malignancy in the mouse, even in the absence of a thymus, parallels the development of B- and T-cell lymphoma and leukemia in the few rare mismatch repair-null human patients that have been identified. The persistent development of hematologic malignancy both in the mouse and in human patients deficient in mismatch repair leads us to implicate mismatch repair as an important repair mechanism in normal B- and T-cell development. Thus, mismatch repair-deficient mice may prove to be a good model to study human hematologic malignancy.     

The DNA mismatch repair genes Msh3 and Msh6 cooperate in intestinal tumor suppression

Repair of mismatches in DNA in mammalian cells is mediated by a complex of proteins that are members of two highly conserved families of genes referred to as MutS and MutL homologues. Germline mutations in several members of these families, MSH2, MSH6, MLH1, and PMS2, but not MSH3, are responsible for hereditary non-polyposis colorectal cancer. To examine the role of MSH3, we generated a mouse with a null mutation in this gene. Cells from Msh3-/- mice are defective in repair of insertion/ deletion mismatches but can repair base-base mismatches. Msh3-/- mice develop tumors at a late age. When the Msh3-/- and Msh6-/- mutations are combined, the tumor predisposition phenotype is indistinguishable from Msh2-/- or Mlh1-/- mice. These results suggest that MSH3 cooperates with MSH6 in tumor suppression.     

Mutational-reporter transgenes rescued from mice lacking either Mgmt, or both Mgmt and Msh6 suggest that O6-alkylguanine-induced miscoding does not contribute to the spontaneous mutational spectrum

O6-methylguanine methyltransferase, Mgmt, constitutes the first line of defense against O6-alkylguanine, which can result in G : C to A : T transitions upon DNA replication. Mgmt has been found in organisms as diverse as archaebacteria and mammals. This evolutionary conservation suggests that all organisms may be exposed to either endogenous or environmental alkylating agents. We thus hypothesized that tissues of Mgmt-/- mice would exhibit elevated mutant frequencies. Employing the Big Blue trade mark transgenic system, we evaluated lacI mutants rescued from liver and small intestinal DNA of young Mgmt-/- mice. Interestingly, while there was a small difference between Mgmt-/- mice and controls with respect to lacI mutant frequency, no differences attributable to Mgmt deficiency were apparent in the mutational spectra. Although mutations stemming from O6-guanine alkylations would be predicted to be cumulative, we found no evidence of an Mgmt-dependent alteration in mutation spectrum in DNA samples from 12 month-old mice. To optimize our ability to detect mutations resulting from O6-alkylguanine-induced G : T mismatches, mice with combined deficiencies of Mgmt and the DNA mismatch repair molecule, Msh6, were analysed. In spite of this strategy, we observed no significant differences between Mgmt-/- Msh6-/- and Msh6-/- mouse lacI mutations, except for a trend towards a greater percentage (of total transitions) of G : C to A : T changes in Mgmt-/-Msh6-/- livers. Therefore, despite the striking evolutionary conservation of Mgmt, deficiency of this gene did not significantly impact the spontaneous lacI mutational spectrum in vivo.     

Mice over-expressing human O6 alkylguanine-DNA alkyltransferase selectively reduce O6 methylguanine mediated carcinogenic mutations to threshold levels after N-methyl-N-nitrosourea.

While it is well known that MNU induces thymic lymphomas in the mouse, it remains unclear which pre-mutagenic lesions are responsible for lymphomagenic transformation. One lesion thought to play a critical role is O6methylguanine[O6mG]which initiates G: C to A:T transition mutations in K-ras and other oncogenes. O6alkylguanine-DNA alkyltransferase (AGT), encoded by the methylguanine methyltransferase gene [MGMT], removes the methyl group thereby preventing the mutation from occurring. When overexpressed in the thymus, MGMT protects mice from MNU-induced thymic lymphomas. To determine whether MGMT overexpression reduced G: C to A: T mutation frequency after MNU, Big Blue lacI and MGMT+/Big Blue mice were treated with MNU and analysed for mutations in the lacI and K-ras genes. The incidence of MNU-induced lymphomas was 84% in Big Blue lacI mice compared to 14% in MGMT+Big Blue lacI mice. Sixty-two per cent of the lymphomas had a GGT to GAT activating mutation in codon 12 of K-ras consistent with O6mG adduct-mediated point mutagenesis. LacI mutation frequency in thymus of MNU treated Big Blue mice was 45-fold above background whereas it was 11-fold above background in MNU treated MGMT+/Big Blue mice. Most lacI mutations were G:C to A:T transitions, implicating O6mG even in the MGMT+mice. No mutations were attributable to chromosomal aberrations or rearrangements. Thus, O6mG adducts account for the carcinogenic effect of MNU and MGMT overexpression is selectively able to reduce O6methylguanine adducts below a carcinogenic threshold. Other adducts are mutagenic but appear to contribute much less to malignant transformation or oncogene activation.     

Msh-2 suppresses in vivo mutation in a gene dose and lesion dependent manner.

Mice deficient for the mismatch repair (MMR) gene Msh2 show accelerated tumourigenesis and a reduced apoptotic response to DNA damage of methylation type. Here we examine the effect of mutation for Msh2 on in vivo mutation frequencies in the intestine as determined by loss of function at the Dolichos biflorus (Dlb-1) locus. Spontaneous mutation frequencies were scored in cohorts of ageing mice either wild type or mutant for Msh2. In mice less than 1 year old, mutation frequencies were only elevated in Msh2 null mice. However, beyond this age heterozygous Msh2 mice showed significantly higher mutation frequencies than controls. These findings implicate a gene dose dependent requirement for Msh2 in mutation suppression and prompted an analysis of young Msh2 mutants following exposure to DNA damage. Following exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), Msh2 deficient mice show a reduced apoptotic response and an increase in mutation frequency. Heterozygotes did not differ from controls. Following exposure to cisplatin, no significant elevation was seen in mutation frequencies, even within homozygotes. This is particularly surprising given the association between cisplatin resistance and MMR deficiency. These findings therefore demonstrate a complex reliance upon functional Msh2 in mutation surveillance. We have identified three separate scenarios. First, where retention of both Msh2 alleles over an extended period of time appears critical to the suppression of spontaneous mutation; second, 3 weeks following exposure to MNNG, where only complete loss of Msh2 results in elevated mutation; and finally following cisplatin exposure, where induced levels of mutation are independent of Msh2 status.     

Msh2 deficiency does not contribute to cisplatin resistance in mouse embryonic stem cells

Several reports have suggested that a defect in the DNA mismatch repair (MMR) system not only causes resistance to methylating agents but also confers low-level resistance to the chemotherapeutic drug cisplatin. Here we report that in a clonogenic assay, mouse embryonic stem (ES) cells deficient for the MMR protein MSH2 respond similarly as wild-type cells to cisplatin. Furthermore, restoring MSH2 expression in a cisplatin-resistant subclone selected from an Msh2(-/-) cell population did not sensitize cells to cisplatin. To ascertain that our observations were not the result of a mutation in the Msh2(-/-) cells that obscured the contribution of a defective MMR machinery to cisplatin resistance, we made use of the Cre-lox system to create a cell line in which the Msh2 gene can be conditionally inactivated. However, while de novo inactivation of Msh2 rendered cells tolerant to the methylating drug N-methyl-N'-nitro-N-nitrosoguanidine as expected, it did not alter the sensitivity to cisplatin. In addition, we were not able to derive cisplatin-resistant subclones from this freshly generated MMR-deficient cell line. Thus, in ES cells we did not find evidence for direct involvement of MMR deficiency in cisplatin resistance.     

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 tumorigenesis.

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(-/-)Apc(1638N) 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 beta-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 beta-catenin regulation. Our analysis therefore has revealed distinct mutational spectra and clarified the roles of Msh3 and Msh6 in DNA repair and intestinal tumorigenesis.     

Cell lineage-specific effects associated with multiple deficiencies of tumor susceptibility genes in Msh2(-/-)Rb(+/-) mice

Cooperative effects of genetic alterations are frequently observed during carcinogenesis.Mice carrying germ-line mutations in both Rb and p53 or Msh2 and p53 die earlier of tumors than mice with only one of these genes inactivated. Mice with a single wild-type Rb allele develop a syndrome of multiple neuroendocrine neoplasia, and inactivation of both alleles of Msh2 gene predisposes mice to gastrointestinal cancer, lymphomas and tumors of the skin that exhibit a mismatch repair defect. Here we showed that Msh2(-/-)Rb(+/-) mice developed lymphomas later than Msh2-deficient littermates, and the lymphomas observed in Msh2(-/-)Rb(+/-) mice have increased rates of apoptosis and rarely spread to other organs and tissues. In contrast to lymphomagenesis, courses of neuroendocrine, intestinal, and skin carcinogenesis were not significantly influenced by the Msh2(-/-)Rb(+/-) genetic combination. In these mice, neuroendocrine tumors displayed a loss of the remaining wild-type Rb allele but did not show microsatellite instability. On the other hand, the intestinal and skin tumors exhibited microsatellite instability but kept the remaining wild-type allele of Rb. Taken together, these data not only revealed a novel biological interaction between Rb and Msh2 but also cell lineage specificity effects associated with multiple deficiencies in these tumor susceptibility genes.     

Msh2 deficiency increases susceptibility to benzo[a]pyrene-induced lymphomagenesis

DNA mismatch repair (MMR) is essential for repair of single-base mismatches and insertion/deletion loops. MMR proteins also participate in cellular response to DNA damaging agents such as various alkylating agents. Mice deficient in the MMR gene Msh2 develop tumors earlier after exposure to alkylating agents when compared to unexposed mice. The interaction between the MMR system and polycyclic aromatic hydrocarbons such as benzo[a]pyrene (B[a]P) has not been investigated in vivo. Here, we show that treatment of Msh2-deficient mice with B[a]P enhances susceptibility to lymphomagenesis. Carrying at least one intact copy of the Msh2 gene had a protective effect. B[a]P treatment only induced lymphomas in 3 of the 40 (7.5%) mice with at least one intact copy of the Msh2 gene as compared to 13 of the 17 (76.5%) Msh2-deficient mice and occurs only after a much longer time period. The B[a]P-DNA adduct levels measured in lung, liver, spleen and forestomach of B[a]P-treated Msh2-/- mice were not significantly different from B[a]P-treated Msh2+/+ mice. In summary, the results suggest that B[a]P accelerates lymphomagenesis in Msh2-deficient mice. Furthermore, Msh2 deficiency does not have any significant effect on B[a]P-DNA adduct levels.     

Selective radiosensitization of drug-resistant MutS homologue-2 (MSH2) mismatch repair-deficient cells by halogenated thymidine (dThd) analogues: Msh2 mediates dThd analogue DNA levels and the differential cytotoxicity and cell cycle effects of the dThd analogues and 6-thioguanine

Mismatch repair (MMR) deficiency, which underlies hereditary nonpolyposis colorectal cancer, has recently been linked to a number of sporadic human cancers as well. Deficiency in this repair process renders cells resistant to many clinically active chemotherapy agents. As a result, it is of relevance to find an agent that selectively targets MMR-deficient cells. We have recently shown that the halogenated thymidine (dThd) analogues iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd) selectively target MutL homologue-1 (MLH1)-deficient human cancer cells for radiosensitization. The levels of IdUrd and BrdUrd in cellular DNA directly correlate with the ability of these analogues to increase the sensitivity of cells and tissues to ionizing radiation, and data from our laboratory have demonstrated that MLH1-mediated MMR status impacts dThd analogue DNA levels, and consequently, analogue-induced radiosensitization. Here, we have extended these studies and show that, both in human and murine cells, MutS homologue-2 (MSH2) is also involved in processing dThd analogues in DNA. Using both E1A-transformed Msh2+/+ and Msh2-/- murine embryonic stem (ES)-derived cells (throughout this report we use Msh2+/+ and Msh2-/- to refer to murine ES-derived cell lines that are wild type or mutant, respectively, for the murine Msh2 gene) and human endometrial cancer cells differing in MSH2 status, we see the classic cytotoxic response to 6-thioguanine (6-TG) in Msh2+/+ and human HEC59/2-4 (MSH2+) MMR-proficient cells, whereas Msh2-/- cells and human HEC59 (MSH2-/-) cells are tolerant (2-log difference) to this agent. In contrast, there is very little cytotoxicity in Msh2+/+ ES-derived and HEC59/2-4 cells to IdUrd, whereas Msh2-/- and HEC59 cells are more sensitive to IdUrd. High-performance liquid chromatography analysis of IdUrd and BrdUrd levels in DNA suggests that this differential cytotoxicity may be due to lower analogue levels in MSH2+ murine and human tumor cells. The DNA levels of IdUrd and BrdUrd continue to decrease over time in Msh2+/+ cells following incubation in drug-free medium, whereas they remain high in Msh2-/- cells. This trend was also found in MSH2-deficient human endometrial cancer cells (HEC59) when compared with HEC59/2-4 (hMsh2-corrected) cells. As a result of higher analogue levels in DNA, Msh2-/- cells are selectively targeted for radiosensitization by IdUrd. Fluorescence-activated cell-sorting analysis of Msh2+/+ and Msh2-/- cells shows that selective toxicity of the halogenated nucleotide analogues is not correlated with a G2-M cell cycle arrest and apoptosis, as is found for selective killing of Msh2+/+ cells by 6-TG. Together, these data demonstrate MSH2 involvement in the processing of IdUrd and BrdUrd in DNA, as well as the differential cytotoxicity and cell cycle effects of the halogenated dThd analogues compared with 6-TG. Therefore, IdUrd and BrdUrd may be used clinically to selectively target both MLH1- and MSH2-deficient, drug-resistant cells for radiosensitization.     

Susceptibility of Msh2-deficient mice to inflammation-associated colorectal tumors

Patients with longstanding extensive ulcerative colitis have an increased risk of developing colorectal cancer (CRC). There are significant differences in the early pathogenesis of colitis-associated tumors compared with common CRC, whereas the frequency, degree, and significance of microsatellite instability (MSI) as a marker of mismatch repair deficiency in colitis tumors remain unclear. Here we describe the application of the DSS model of chronic colitis to mice with a defect in the Msh2 mismatch repair gene to discern these early events. These mice do not develop CRC spontaneously without an external trigger. The aim of this study was to determine the effect of the Msh2 defect on the frequency and grade of colitis-associated colorectal dysplasia and adenocarcinoma in Msh2-/-, Msh2+/-, and wild-type (Msh2+/+) mice and on the MSI status of the tumors. We show that in mice with chronic colitis, 60% of the Msh2-/- and 29% of the wild-type mice developed high-grade dysplasia or adenocarcinoma, but heterozygosity for the Msh2 defect did not increase tumor susceptibility over wild-type genotype. The largest difference between genotypes was in the frequency of high-grade dysplasia, with 46.7, 8, and 12.5% in Msh2-/-, Msh2+/-, and Msh2+/+ mice, respectively. The Msh2-/- mice developed MSI-high tumors, whereas the majority of the Msh2+/- and wild-type tumors had no MSI. In the Msh2-/- mice, MSI appeared early in non-neoplastic colon tissue, presumably as a result of markedly increased epithelial cell proliferation associated with inflammation. These observations suggest that a homozygous mismatch repair defect predisposes to tumors triggered by chronic inflammation but is not the only factor involved because tumors also developed in the wild-type mice. This model of colitis offers opportunities to characterize the different molecular pathways of carcinogenesis operating in chronic colitis.     

Increased incidence of endometrioid tumors caused by aberrations in E-cadherin promoter of mismatch repair-deficient mice

Loss of E-cadherin expression is a critical step in the development and progression of gynecological tumors. Study of the precise role of E-cadherin has been hampered by the lack of satisfactory mouse model for E-cadherin deficiency. Likewise, DNA mismatch repair (MMR) is implicated in gynecological tumorigenesis, but knockout of MMR in mice predominantly causes hematologic neoplasms. Here, we show that combined disruption of E-cadherin and DNA MMR pathways increases incidence of endometrioid tumors in mice. Twenty percent of mice knockout for Msh2 enzyme and hemizygous for E-cadherin [Msh2(-/-)/Cdh1(+/-)] developed endometrioid-like tumors in the ovary, uterus and genital area. Characteristic of these tumors was a complete loss of E-cadherin expression. Sequence analysis of E-cadherin promoter region demonstrated that the loss of E-cadherin expression is caused by inactivating mutations, implying that E-cadherin is a mutational target in Msh2-deficient mice. In addition, Msh2(-/-)/Cdh1(+/-) mice showed a reduction in overall survival as compared with their Msh2(-/-) counterparts due to the development of more aggressive lymphomas, suggesting a specific role of E-cadherin in lymphomagenesis. In conclusion, Msh2(-/-)/Cdh1(+/-) mice provide a good model of gynecological tumorigenesis and may be useful for testing molecular target-specific therapies.