The absence of Msh2 alters abelson virus pre-B-cell transformation by influencing p53 mutation.

Defects in DNA mismatch repair predispose cells to the development of several types of malignant disease. The absence of Msh2 or Mlh1, two key molecules that mediate mismatch repair in eukaryotic cells, increases the frequency of mutation and also alters the response of some cells to apoptosis and cell cycle arrest. To understand the way these changes contribute to cancer predisposition, we examined the effects of defective mismatch repair on the multistep process of pre-B-cell transformation by Abelson murine leukemia virus. In this model, primary transformants undergo a prolonged apoptotic crisis followed by the emergence of fully transformed cell lines. The latter event is correlated to a loss of function of the p53 tumor suppressor protein and down-modulation of the p53 regulatory protein p19Arf. Analyses of primary transformants from Msh2 null mice and their wild-type littermates revealed that both types of cells undergo crisis. However, primary transformants from Msh2 null animals recover with accelerated kinetics, a phenomenon that is strongly correlated to the appearance of cells that have lost p53 function. Analysis of the kinetics with which p53 function is lost revealed that this change provides the dominant stimulus for emergence from crisis. Therefore, the absence of mismatch repair alters the molecular mechanisms involved in transformation by affecting a gene that controls apoptosis and cell cycle progression, rather than by affecting these processes directly.

Tissues of MSH2-deficient mice demonstrate hypermutability on exposure to a DNA methylating agent.

The mutational response of mismatch repair-deficient animals to the alkylating agent N-methyl-N-nitrosourea was evaluated by using a transgenic lacI reporter system. Although the mutations detected in MSH2 heterozygotes were similar to those of controls, MSH2-/- animals demonstrated striking increases in mutation frequency in response to this agent. G:C to A:T transitions at GpG sites, as opposed to CpG sites, dominated the mutational spectrum of both MSH2+/+ and MSH2-/- N-methyl-N-nitrosourea -treated animals. Extrapolating to humans with hereditary non-polyposis colorectal cancer, the results suggest that MSH2 heterozygotes are unlikely to be at increased risk of mutation, even when exposed to potent DNA methylating agents. In contrast, mismatch repair-deficient cells spontaneously arising within individuals with hereditary non-polyposis colorectal cancer would likely exhibit hypermutability in response to such mutagens, an outcome predicted to accelerate the pace of tumorigenesis.

Mutator phenotype in Msh2-deficient murine embryonic fibroblasts.

Embryonic fibroblast cell lines were established from mice deficient, heterozygous, or proficient for Msh2, one of the three known DNA mismatch repair genes involved in hereditary nonpolyposis colon cancer (HNPCC). Cell lines were established by transfection of primary mouse embryo fibroblasts with E7 and Ras oncogenes or mutant p53. Spontaneously immortalized cells derived from the primary cultures were also studied. To determine whether these cells developed a mutator phenotype similar to that found in colon cancer cells deficient in mismatch repair, we measured mutation rates, microsatellite instability, and sensitivities to a range of DNA-damaging agents. The mutator phenotype detected in the E7 and Ras or mutant p53-immortalized Msh2-/- mouse cells was similar to that found in human mismatch repair-deficient colorectal carcinoma cell lines. Mutation rates to ouabain resistance were increased 8-12-fold relative to lines from Msh2+/+ mice, and microsatellite instability was detectable in 12-18% of subclones derived from the Msh2-/- line but was undetectable in subclones developed from the Msh2+/+ line. Furthermore, E7 and Ras or spontaneously immortalized Msh2-/- cells were significantly more resistant to the cytotoxic effects of 6-thioguanine relative to Msh2+/+ cells. In contrast, these lines showed various responses to UV light and cis-platinum, suggesting that mismatch repair deficiency was not the sole determinant for sensitivity to these DNA-damaging agents. Particular attention was paid to the properties of cells heterozygous for the Msh2 mutant gene, which would mimic the situation of an HNPCC carrier. However, our studies failed to reveal any properties of these cells that might provide a growth advantage or predispose them for the acquisition of further mutations. This observation is consistent with the model that inactivation of the wild-type Msh2 allele is a critical step for tumorigenesis in HNPCC patients.

Base transitions dominate the mutational spectrum of a transgenic reporter gene in MSH2 deficient mice.

Tumors derived from individuals with hereditary nonpolyposis colorectal cancer syndrome frequently demonstrate mutations in both alleles of hMSH2, a key gene in DNA mismatch repair (MMR). Sporadic tumors also frequently exhibit MMR deficiency. In keeping with the role of MMR in the maintenance of genome integrity, mice deficient in MSH2 via gene targeting demonstrate a high incidence of thymic lymphomas and small intestinal adenocarcinomas. To investigate the effects of MSH2 deficiency in normal tissues, mice containing a retrievable transgenic lacI reporter gene for mutation detection were crossed with MSH2-/- mice. Mice homozygous for MSH2 deficiency revealed 4.8, 11.0 and 15.2-fold elevations in spontaneous mutation frequency in DNA obtained from brain, small intestine, and thymus, respectively, as compared to heterozygous or wild-type mice. Mutations most frequently recovered from MSH2-/- mice were single base substitutions (77%), particularly base transitions (64%). Frameshifts occurred less frequently (19%) and fell within very short (3-5 bp) mononucleotide runs. Thus the number of key growth control genes potentially impacted by MMR deficiency extends beyond those containing repetitive sequences. These results highlight the capacity for MSH2 deficiency to serve as a potent driving force during the multi-step evolution of tumors.

Defects of the mismatch repair gene MSH2 are implicated in the development of murine and human lymphoblastic lymphomas and are associated with the aberrant expression of rhombotin-2 (Lmo-2) and Tal-1 (SCL).

Mutations in the DNA mismatch repair (MMR) gene hMSH2 underlie a novel pathway of tumorigenesis for some cancers of epithelial origin. Mice deficient in MSH2 are susceptible to lymphomas but defects in this gene have not been identified in human lymphoid tumors. To determine if the lymphomas these mice develop are related to a particular subtype of human lymphoma we evaluated 20 clinically ill homozygous MSH2-/- mice ranging in age from 2 to 13 months. The murine tumors comprised a single histopathologic entity representing the malignant counterpart of precursor thymic T cells and closely resembled human precursor T-cell lymphoblastic lymphoma (LBL). Evaluation of the expression of three T-cell malignancy associated genes showed that Rhombotin-2 (RBTN-2 also known as Lmo-2), TAL-1 (also known as SCL), and HOX-11 were expressed in 100%, 40%, and 0% of the murine tumors, respectively. The MSH2-/- murine model of precursor T-cell LBL was substantiated by the finding of a nearly identical expression profile of RBTN-2, TAL-1, and HOX-11 in 10 well-characterized cases of human LBL. Direct evidence for MSH2 abnormalities in human LBL was established by sequence analysis of exon 13 of hMSH2, which revealed coding region mutations in 2 of 10 cases. Our findings implicate defects in the MMR system with the aberrant expression of T-cell specific proto-oncogenes and define a new pathway of human lymphomagenesis.

Spontaneous intestinal carcinomas and skin neoplasms in Msh2-deficient mice.

Hereditary nonpolyposis colorectal cancer is associated with defects in DNA mismatch repair. Here, we characterize tumor susceptibility of the recently described Msh2-deficient mouse model. Within the first year of observation, all homozygous mice succumbed to disease, with lymphomas observed in at least 80% of the cases. The majority (70%) of animals 6 months or older developed intestinal neoplasms associated with APC inactivation. Microsatellite instability was more common in carcinomas than in adenomas, but uncommon in normal tissues. Some animals (7%) developed a variety of skin neoplasms analogous to the Muir-Torre syndrome. Msh2-/- mice implicate a direct role for mismatch repair in several neoplasms with striking phenotypic similarities to humans.

MSH2 deficiency contributes to accelerated APC-mediated intestinal tumorigenesis.

Accelerated intestinal tumorigenesis is probable in hereditary nonpolyposis colorectal cancer, a condition associated with germ line DNA mismatch repair (MMR) gene defects, and is believed to be caused by rapid accumulation of replication errors in critical genes, such as the APC (adenomatous polyposis coli) tumor suppressor gene. To study the potential contribution of MMR genes to accelerated intestinal tumorigenesis, we crossed the Min mouse, heterozygous for a germ line mutation of Apc, with an MMR gene (Msh2)-deficient mouse. MSH2 deficiency resulted in the development of many colonic aberrant crypt foci, as well as reduced survival of the mice, secondary to both a greater number and more rapidly developing adenomas. The mechanism of inactivation of the wild-type Apc allele depended on MSH2 status. In the presence of functional MSH2, all tumors demonstrated loss of heterozygosity. In contrast, whereas all adenomas were APC negative by immunostaining, only 5 of 34 adenomas from Apc+/-/Msh2-/- mice demonstrated loss of heterozygosity of the wild-type Apc allele, suggesting that somatic Apc mutations are responsible for the additional tumors. These findings provide evidence for the important role of MMR genes in accelerated intestinal tumorigenesis, thus supporting more aggressive surveillance strategies to prevent colorectal cancer in hereditary nonpolyposis colorectal cancer.

MSH2 deficient mice are viable and susceptible to lymphoid tumours.

Alterations of the human MSH2 gene, a homologue of the bacterial MutS mismatch repair gene, co-segregate with the majority of hereditary non-polyposis colon cancer (HNPCC) cases. We have generated homozygous MSH2-/- mice. Surprisingly, these mice were found to be viable, produced offspring in a mendelian ratio and bred through at least two generations. Starting at two months of age homozygous-/- mice began, with high frequency, to develop lymphoid tumours that contained microsatellite instabilities. These data establish a direct link between MSH2 deficiency and the pathogenesis of cancer. These mutant mice should be good models to study the progression of tumours and also to screen carcinogenic and anti-cancer agents.