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PubMed2001

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.

Animals↗
PubMed2002

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.

Adenocarcinoma↗
PubMed2002

MSH2 in contrast to MLH1 and MSH6 is frequently inactivated by exonic and promoter rearrangements in hereditary nonpolyposis colorectal cancer.

To estimate the relative frequency of mismatch repair genes, rearrangements in hereditary nonpolyposis colorectal cancer (HNPCC) families without detectable mutations in MSH2 or MLH1, we have analyzed by multiplex PCR of short fluorescent fragments MSH2, MLH1, and MSH6 in 61 families, either fulfilling Amsterdam criteria or including cases of multiple primary cancers belonging to the HNPCC spectrum. We detected 13 different genomic rearrangements of MSH2 in 14 families (23%), whereas we found no rearrangement of MLH1 and MSH6. Analysis of 31 other families, partially meeting Amsterdam criteria, revealed no additional rearrangement of MSH2. All of the MSH2 rearrangements, except one, corresponded to genomic deletions involving one or several exons. In 8 of 13 families with a MSH2 genomic deletion, the MSH2 promoter was also deleted, and the 5' breakpoint was located either within or upstream the MSH2 gene. This study demonstrates the heterogeneity of MSH2 exonic and promoter rearrangements and shows that, in HNPCC families without detectable MSH2 or MLH1 point mutation, one must consider the presence of MSH2 genomic rearrangements before the involvement of other mismatch repair genes. The simplicity and rapidity of their detection, using fluorescent multiplex PCR, led us to recommend to begin the molecular analysis in HNPCC by screening for MSH2 rearrangements.

Adaptor Proteins, Signal Transducing↗
PubMed2003

The mammalian mismatch repair protein MSH2 is required for correct MRE11 and RAD51 relocalization and for efficient cell cycle arrest induced by ionizing radiation in G2 phase.

In yeast, MSH2 plays an important role in mismatch repair (MMR) and recombination, whereas the function of the mammalian MSH2 protein in recombinational repair is not completely established. We examined the cellular responses of MSH2-deficient mouse cells to X-rays to clarify the role of MSH2 in recombinational repair. Cell survival, checkpoint functions and relocalization of the recombination-related proteins MRE11 and RAD51 were analysed in embryonic fibroblasts derived from MSH2(+/+) and MSH2(-/-) mice, and in MSH2-proficient and deficient mouse colorectal carcinoma cells. Loss of MSH2 function was found to be associated with reduction in cell survival following radiation, absence of either MRE11 or RAD51 relocalization and a higher level of X-ray-induced chromosomal damage specifically in G2-phase cells. Finally, MSH2(-/-) cells showed an inefficient early G2/M checkpoint, being arrested only transiently after irradiation before progressing into mitosis. Consistent with the premature release from the G2-phase arrest, activation of CHK1 was transient and CHK2 was not phosphorylated in synchronized MSH2-null cells. Our data suggest that an active MSH2 is required for a correct response to ionizing radiation-induced DNA damage in the G2 phase of the cell cycle, possibly connecting DSB repair to checkpoint signalling.

Animals↗
PubMed2000

Genetic analysis of mouse embryonic stem cells bearing Msh3 and Msh2 single and compound mutations.

We have previously described the use of homologous recombination and CRE-loxP-mediated marker recycling to generate mouse embryonic stem (ES) cell lines homozygous for mutations at the Msh3, Msh2, and both Msh3 and Msh2 loci (2). In this study, we describe the analysis of these ES cells with respect to processes known to be affected by DNA mismatch repair. ES cells homozygous for the Msh2 mutation displayed increased resistance to killing by the cytotoxic drug 6-thioguanine (6TG), indicating that the 6TG cytotoxic mechanism is mediated by Msh2. The mutation rate of the herpes simplex virus thymidine kinase 1 (HSV-tk1) gene was unchanged in Msh3-deficient ES cell lines but markedly elevated in Msh2-deficient and Msh3 Msh2 double-mutant cells. Notably, the HSV-tk1 mutation rate was 11-fold higher, on average, than that of the hypoxanthine-guanine phosphoribosyl transferase (Hprt) locus in Msh2-deficient cells. Sequence analysis of HSV-tk1 mutants from these cells indicated the presence of a frameshift hotspot within the HSV-tk1 coding region. Msh3-deficient cells displayed a modest (16-fold) elevation in the instability of a dinucleotide repeat, whereas Msh2-deficient and Msh2 Msh3 double-mutant cells displayed markedly increased levels of repeat instability. Targeting frequencies of nonisogenic vectors were elevated in Msh2-deficient ES cell lines, confirming the role of Msh2 in blocking recombination between diverged sequences (homeologous recombination) in mammalian cells. These results are consistent with accumulating data from other laboratories and support the current model of DNA mismatch repair in mammalian cells.

Animals↗
PubMed1997

Differential effects of the mismatch repair genes MSH2 and MSH3 on homeologous recombination in Saccharomyces cerevisiae.

The products of the yeast mismatch repair genes MSH2 and MSH3 participate in the inhibition of genetic recombination between homeologous (divergent) DNA sequences. In strains deficient for these genes, homeologous recombination rates between repeated elements are elevated due to the loss of this inhibition. In this study, the effects of these mutations were further analyzed by quantitation of mitotic homeologous recombinants as crossovers, gene conversions or exceptional events in wild-type, msh2, msh3 and msh2 msh3 mutant strains. When homeologous sequences were present as a direct repeat in one orientation, crossovers and gene conversions were elevated in msh2, msh3 and msh2 msh3 strains. The increases were greater in the msh2 msh3 double mutant than in either single mutant. When the order of the homeologous sequences was reversed, the msh2 mutation again yielded increased rates of crossovers and gene conversions. However, in an msh3 strain, gene conversions occurred at higher levels but interchromosomal crossovers were not increased and intrachromosomal crossovers were reduced relative to wild type. The msh2 msh3 double mutant behaved like the msh2 single mutant in this orientation. Control strains harboring homologous duplications were largely but not entirely unaffected in mutant strains, suggesting specificity for the mismatched intermediates of homeologous recombination. In all strains, very few (< 10%) recombinants could be attributed to exceptional events. These results suggest that MSH2 and MSH3 can function differentially to control homeologous exchanges.

DNA Repair↗
PubMed2003

Msh2 ATPase activity is essential for somatic hypermutation at a-T basepairs and for efficient class switch recombination.

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytidine deaminase-mediated cytidine deamination of immunoglobulin genes. MutS homologue (Msh) 2-/- mice have reduced A-T mutations and CSR. This suggests that Msh2 may play a role in repairing activation-induced cytidine deaminase-generated G-U mismatches. However, because Msh2 not only initiates mismatch repair but also has other functions, such as signaling for apoptosis, it is not known which activity of Msh2 is responsible for the effects observed, and consequently, many models have been proposed. To further dissect the role of Msh2 in SHM and CSR, mice with a "knockin" mutation in the Msh2 gene that inactivates the adenosine triphosphatase domain were examined. This mutation (i.e., Msh2G674A), which does not affect apoptosis signaling, allows mismatches to be recognized but prevents Msh2 from initiating mismatch repair. Here, we show that, similar to Msh2-/- mice, SHM in Msh2G674A mice is biased toward G-C mutations. However, CSR is partially reduced, and switch junctions are more similar to those of postmeiotic segregation 2-/- mice than to Msh2-/- mice. These results indicate that Msh2 adenosine triphosphatase activity is required for A-T mutations, and suggest that Msh2 has more than one role in CSR.

Adenosine Triphosphatases↗
PubMed2001

1,2-Dimethylhydrazine-induced colon carcinoma and lymphoma in msh2(-/-) mice.

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.

1,2-Dimethylhydrazine↗
PubMed2004

Microtubule stress modifies intra-nuclear location of Msh2 in mouse embryonic fibroblasts.

The maintenance of genomic stability in mitotic and meiotic cycles through mismatch repair (MMR) demands the coordination of MMR functions with multiple processes including cell cycle traverse, linked changes in microtubule dynamics, protein translocation at chromatin sites and checkpoint activation. We have studied changes in the intracellular location of the MMR protein Msh2 in response to mitosis, microtubule disruption by colcemid and DNA damage induction by cis-platin in mouse embryonic fibroblasts (MEFs). Image analysis indicated that MEFs have a normally high nuclear retention of Msh2 during interphase with a precipitous dispersal of protein from chromatin sites into the cytoplasm at mitosis. Dispersal was also observed in cisplatin- and colcemid-treated interphase MEFs without any change in the overall Msh2 levels throughout the cell cycle. There was no evidence of co-localization of the punctate cytoplasmic Msh2 foci with any microtubule structures and knockout of Msh2 altered neither the extent of microtubule disruption nor the functional activation of the spindle assembly checkpoint by colcemid. Critically, extra-nuclear relocation of protein did not alter the ability to mount an Msh2-dependent G2 checkpoint delay in response to cisplatin-induced DNA damage. Depletion of the nuclear pool of Msh2 protein in cells undergoing dispersal was found to involve a rapid relocation of protein from AT-rich chromatin sites as defined by coassociation studies exploiting a newly-characterized base-pair preference of the fluorescent DNA binding probe DRAQ5. The study reveals the unexpected mobility of MMR protein pools during the MEF cell cycle and in response to different stress-inducing agents. The results link for the first time microtubule-integrity with intra-nuclear Msh2 protein dynamics. The high nuclear retention of Msh2 in interphase MEFs is in contrast to human tumor cells while the observations on protein dispersal suggest that only low levels of nuclear-located Msh2 are needed for G2 checkpoint activation by DNA damage.

Animals↗
PubMed2006

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.

Animals↗
PubMedDate not supplied

Cumulative incidence of colorectal and extracolonic cancers in MLH1 and MSH2 mutation carriers of hereditary nonpolyposis colorectal cancer.

The extracolonic tumor spectrum of hereditary nonpolyposis colorectal cancer (HNPCC) includes cancer of the endometrium, ovaries, stomach, biliary tract, and urinary tract. This study was designed to determine the penetrance of colorectal and extracolonic tumors in HNPCC mutation carriers. Forty-nine patients (22 females and 27 males) were identified with an MSH2 germline mutation, and 56 patients (28 females and 28 males) were identified with an MLH1 I mutation. Cumulative incidence by age 60 (lifetime risk) and mean age of cancer diagnosis were compared. The lifetime risk of extracolonic cancers in MSH2 and MLH1 carriers was 48% and 11%, respectively (P = 0.016). Extracolonic cancer risk in MSH2 females and males was 69% and 34%, respectively (P = 0.042). Mean age of extracolonic cancer diagnosis was significantly older for MSH2 males than females (55.4 vs. 39.0, P = 0.013). No difference was observed in colorectal cancer risk between MLH1 and MSH2 carriers (84% vs. 71%). Colorectal cancer risk was 96% in MSH2 males compared to 39% in MSH2 females (P = 0.034). No differences in colorectal and extracolonic cancer risks between MLH1 females and males were identified. The risk of extracolonic cancer by age 60 was greater in MSH2 mutation carriers than in MLH1 carriers. Gender differences in colorectal and extracolonic cancer risk were observed for MSH2 carriers only. These phenotypic features of HNPCC genotypes may have clinical significance in the design of genotype-specific screening, surveillance, and follow-up for affected individuals.

Adaptor Proteins, Signal Transducing↗
PubMed2005

The role of MLH1, MSH2 and MSH6 in the development of multiple colorectal cancers.

There is increased incidence of microsatellite instability (MSI) in patients who develop multiple primary colorectal cancers (CRC), although the association with hereditary nonpolyposis colon cancer (HNPCC) is unclear. This study aims to evaluate the underlying genetic cause of MSI in these patients. Microsatellite instability was investigated in 111 paraffin-embedded CRCs obtained from 78 patients with metachronous and synchronous cancers, and a control group consisting of 74 cancers from patients with a single CRC. Tumours were classified as high level (MSI-H), low level (MSI-L) or stable (MSS). MLH1, MSH2 and MSH6 gene expression was measured by immunohistochemistry. Methylation of the MLH1 promoter region was evaluated in MSI-H cancers that failed to express MLH1, and mutational analysis performed in MSI-H samples that expressed MLH1, MSH2 and MSH6 proteins. The frequency of MSI-H was significantly greater in the multiple, 58 out of 111 (52%), compared to the single cancers, 10 out of 74 (13.5%), P < 0.01. Of the 32 patients from whom two or more cancers were analysed, eight (25%) demonstrated MSI-H in both cancers, 13 (41%) demonstrated MSI-H in one cancer and 11 (34%) failed to demonstrate any MSI-H. MSI-H single cancers failed to express MLH1 or MSH2 in seven out of nine (78%) cases and MSI-L/MSS cancers failed to express MLH1 or MSH2 in one out of 45 (2.2%) cases, all cancers expressed MSH6. MSI-H multiple cancers failed to express MLH1 or MSH2 in 21 out of 43 (48%) cases and MSI-L/MSS cancers failed to express MLH1 or MSH2 in four out of 32 (12.5%) cases. MSH6 expression was lost in five MSI-H multiple cancers, four of which also failed to express MLH1 or MSH2. Loss of expression of the same mismatch repair (MMR) gene was identified in both cancers from six out of 19 (31%) patients. Methylation was identified in 11 out of 17 (65%) multiple and three out of six (50%) single MSI-H cancers that failed to express MLH1. Mutational analysis of 10 MSI-H multiple cancers that expressed MLH1, MSH2 and MSH6 failed to demonstrate mutations in the MLH1 or MSH2 genes. We suggest that, although MSI-H is more commonly identified in those with multiple colorectal cancers, this does not commonly arise from a classical HNPCC pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Msh2 status modulates both apoptosis and mutation frequency in the murine small intestine.

Deficiency in genes involved in DNA mismatch repair increases susceptibility to cancer, particularly of the colorectal epithelium. Using Msh2 null mice, we demonstrate that this genetic defect renders normal intestinal epithelial cells susceptible to mutation in vivo at the Dlb-1 locus. Compared with wild-type mice, Msh2-deficient animals had higher basal levels of mutation and were more sensitive to the mutagenic effects of temozolomide. Experiments using Msh2-deficient cells in vitro suggest that an element of this effect is attributable to increased clonogenicity. Indeed, we show that Msh2 plays a role in the in vivo initiation of apoptosis after treatment with temozolomide, N-methyl-N'-nitro-N-nitrosoguanidine, and cisplatin. This was not influenced by the in vivo depletion of O6-alkylguanine-DNA-alkyltransferase after administration of O6-benzylguanine. By analyzing mice mutant for both Msh2 and p53, we found that the Msh2-dependent apoptotic response was primarily mediated through a p53-dependent pathway. Msh2 also was required to signal delayed p53-independent death. Taken together, these studies characterize an in vivo Msh2-dependent apoptotic response to methylating agents and raise the possibility that Msh2 deficiency may predispose to malignancy not only through failed repair of mismatch DNA lesions but also through the failure to engage apoptosis.

Animals↗
PubMed2002

Loss of DNA mismatch repair proteins in skin tumors from patients with Muir-Torre syndrome and MSH2 or MLH1 germline mutations: establishment of immunohistochemical analysis as a screening test.

Muir-Torre syndrome (MTS) is a rare autosomal-dominant disorder characterized by the predisposition to both sebaceous skin tumors (or multiple keratoacanthomas) and internal malignancies. A subtype of MTS is allelic to hereditary nonpolyposis colorectal cancer and is caused by germline mutations in the DNA mismatch repair genes MSH2 or MLH1. In these cases both internal and skin tumors show characteristic microsatellite instability (MSI). The aim of the present study was to determine whether immunohistochemical examination of MSH2 or MLH1 protein expression in MTS-associated skin tumors can be used as a diagnostic screening tool to identify patients with germline mutations in MSH2 or MLH1. In the present study 28 skin lesions from 17 patients (20 sebaceous gland tumors, 4 sebaceous hyperplasias, 3 keratoacanthomas, and 1 squamous cell carcinoma) were tested immunohistochemically with antibodies against MSH2 and MLH1. Eighteen of these tumors were from eight patients with known MSH2 germline mutations, two tumors were from a patient with a germline mutation in MLH1, and eight microsatellite stable sporadic skin tumors served as controls. One sample had to be excluded because of a lack of immunoreactivity. All eight microsatellite stable tumors expressed both DNA repair proteins. In 15 of the tumors from MSH2 germline mutation carriers, loss of MSH2 expression was observed, one tumor showed reduced MSH2 expression, and one tumor displayed positive immunoreactivity to MSH2. Both tumors of the MLH1 germline mutation carrier showed loss of the MLH1 protein. In conclusion, our findings demonstrate that immunohistochemical testing of MTS-related skin tumors is a reliable screening method with high predictive value for the diagnosis of the DNA mismatch repair-deficient MTS.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Evaluation of microsatellite instability and immunohistochemistry for the prediction of germ-line MSH2 and MLH1 mutations in hereditary nonpolyposis colon cancer families.

Forty-eight hereditary nonpolyposis colorectal carcinoma (HNPCC) families for which a tumor sample was available were evaluated for the presence of germ-line mutations in MSH2 and MLH1, tumor microsatellite instability (MSI), and where possible, expression of MSH2 and MLH1 in tumors by immunohistochemistry. Fourteen of 48 of the families had a germ-line mutation in either MSH2 or MLH1 that could be detected by genomic DNA sequencing, and 28 of 48 of the families had MSI-H tumors. Four additional families showed loss of expression of MSH2, and one additional family showed loss of expression of MLH1 but did not have germ-line mutations in MSH2 or MLH1 that could be detected by DNA sequencing. MSI-H, as defined using the National Cancer Institute recommended five-microsatellite panel, had a 100% sensitivity for identifying samples having MSH2 or MLH1 mutations or loss of expression. In contrast, loss of MSH2 and MLH1 expression did not identify all samples having germ-line mutations in MSH2 or MLH1, because in five cases, a mutant protein product was expressed that could be detected by IHC. A combination of the Bethesda criteria for HNPCC and an MSI-H phenotype defined the smallest number of cases having all of the germ-line MSH2 and MLH1 mutations that could be detected by DNA sequencing.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

Mismatch repair protein MSH2 expression and prognosis of colorectal cancer patients.

INTRODUCTION AND AIMS: The role of genetic factors in the etiology and prognosis of patients with sporadic colorectal cancer is controversial. We have therefore investigated the biological and clinicopathological influence of immunohistochemical MSH2 expression in colorectal cancer. PATIENTS AND METHODS: A total of 49 consecutive patients with unselected colorectal cancer operated on in our unit were included in the study. All tumors were resected and tumor specimens were evaluated for MSH2 expression. Clinicopathological data and patient survival were correlated with MSH2 staining. Uni- and multivariate analyses were performed. The minimum follow-up period was five years. RESULTS: Curative resection was performed in 34 patients (64.9%), 14 of whom subsequently relapsed. At the end of the overall follow-up 25 (51%) patients had died, 21 of cancer-related causes. Twenty-eight patients (57.1%) were negative for MSH2 staining. Only vascular invasion was significantly correlated with MSH2 expression (lower median values; p=0.04). The overall median survival was 47.9 months (95% CI=27-86.6%). Multivariate analysis of variables in relation to survival showed that T stage (p=0.001), N stage (p<0.001) and MSH2 expression (p=0.01) were independent factors for survival. CONCLUSIONS: Reduced MSH2 expression is frequent in unselected colorectal cancer patients. Only vascular invasion was correlated with MSH2 expression in this study. Survival was related to TN stage and MSH2 staining.

Adult↗
PubMed1997

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.

Animals↗
PubMed2001

Optimization of experimental conditions for RNA-based sequencing of MLH1 and MSH2 genes.

The most sensitive technique for the detection of germline mutations is exon by exon sequencing of the gene under investigation using genomic DNA as a template for analysis. This approach, however, has cost and sensitivity limitations that can, at least in part, be overcome by RNA-based analysis. Germline mutations of MLH1 and MSH2 are the most frequent cause of the inherited susceptibility to colorectal and other epithelial cancers known as hereditary non-polyposis colorectal cancer (HNPCC). We compared the analysis of the MLH1 and MSH2 genes using mRNA and genomic DNA as starting material from 21 HNPCC patients. All samples were investigated by RT-PCR, sequencing of cDNA and simultaneous sequencing of genomic DNA. The cDNA was generated using specific primers complementary to the ends of MLH1 and MSH2 genes, respectively. Mutations in MLH1 and MSH2 were detected in 11 out of 21 unrelated patients. In 10 out of 11 cases, mutations were detected independently of the type of primers used for reverse transcription (RT). One novel missense mutation (K751R) in MLH1 was detected using this method. One nonsense mutation (E205X) in MSH2 was only detectable when RT was performed using MSH2 gene-specific primers. Shorter PCR products indicative of alternatively spliced transcripts were not observed when MLH1 or MSH2 specific cDNA RT primers were employed to generate template, except in one case where exon skipping was observed for exons 9 and 10. In this report we demonstrate that primers specific for RT of MLH1 and MSH2 are crucial for increasing the sensitivity of cDNA analysis. DNA sequencing using RNA as a basis for template construction may be a valuable and economical alternative to genomic DNA sequencing.

Adaptor Proteins, Signal Transducing↗