Frequency of ApoB and ApoE gene mutations as causes of hypobetalipoproteinemia in the framingham offspring population

Rearranged immunoglobulin variable genes are extensively mutated after stimulation of B lymphocytes by antigen. Mutations are likely generated by an error-prone DNA polymerase, and the mismatch repair pathway may process the mispairs. To examine the role of the MSH2 mismatch repair protein in hypermutation, Msh2-/- mice were immunized with oxazolone, and B cells were analyzed for mutation in their VkappaOx1 light chain genes. The frequency of mutation in the repair-deficient mice was similar to that in Msh2+/+ mice, showing that MSH2-dependent mismatch repair does not cause hypermutation. However, there was a striking bias for mutations to occur at germline G and C nucleotides. The results suggest that the hypermutation pathway frequently mutates G.C pairs, and a MSH2-dependent pathway preferentially corrects mismatches at G and C.     

Severe attenuation of the B cell immune response in Msh2-deficient mice

Recently, results obtained from mice with targeted inactivations of postreplication DNA mismatch repair (MMR) genes have been interpreted to demonstrate a direct role for MMR in antibody variable (V) gene hypermutation. Here we show that mice that do not express the MMR factor Msh2 have wide-ranging defects in antigen-driven B cell responses. These include lack of progression of the germinal center (GC) reaction associated with increased intra-GC apoptosis, severely diminished antigen-specific immunoglobulin G responses, and near absence of anamnestic responses. Mice heterozygous for the Msh2 deficiency display an "intermediate" phenotype in these regards, suggesting that normal levels of Msh2 expression are critical for the B cell response. Interpretation of the impact of an MMR deficiency on the mechanism of V gene somatic hypermutation could be easily confounded by these perturbations.     

Effect of repetitive high-dose treatment with soluble complement receptor type 1 and cobra venom factor on discordant xenograft survival

Mice that are deficient in either the Pms2 or Msh2 DNA mismatch repair genes have microsatellite instability and a predisposition to tumours. Interestingly, Pms2-deficient males display sterility associated with abnormal chromosome pairing in meiosis. Here mice deficient in another mismatch repair gene, Mlh1, possess not only microsatellite instability but are also infertile (both males and females). Mlh1-deficient spermatocytes exhibit high levels of prematurely separated chromosomes and arrest in first division meiosis. We also show that Mlh1 appears to localize to sites of crossing over on meiotic chromosomes. Together these findings suggest that Mlh1 is involved in DNA mismatch repair and meiotic crossing over.     

Mutation in the mismatch repair gene Msh6 causes cancer susceptibility

Mice carrying a null mutation in the mismatch repair gene Msh6 were generated by gene targeting. Cells that were homozygous for the mutation did not produce any detectable MSH6 protein, and extracts prepared from these cells were defective for repair of single nucleotide mismatches. Repair of 1, 2, and 4 nucleotide insertion/deletion mismatches was unaffected. Mice that were homozygous for the mutation had a reduced life span. The mice developed a spectrum of tumors, the most predominant of which were gastrointestinal tumors and B- as well as T-cell lymphomas. The tumors did not show any microsatellite instability. We conclude that MSH6 mutations, like those in some other members of the family of mismatch repair genes, lead to cancer susceptibility, and germline mutations in this gene may be associated with a cancer predisposition syndrome that does not show microsatellite instability.     

Saccharomyces cerevisiae Msh2p and Msh6p ATPase activities are both required during mismatch repair

In the Saccharomyces cerevisiae Msh2p-Msh6p complex, mutations that were predicted to disrupt ATP binding, ATP hydrolysis, or both activities in each subunit were created. Mutations in either subunit resulted in a mismatch repair defect, and overexpression of either mutant subunit in a wild-type strain resulted in a dominant negative phenotype. Msh2p-Msh6p complexes bearing one or both mutant subunits were analyzed for binding to DNA containing base pair mismatches. None of the mutant complexes displayed a significant defect in mismatch binding; however, unlike wild-type protein, all mutant combinations continued to display mismatch binding specificity in the presence of ATP and did not display ATP-dependent conformational changes as measured by limited trypsin protease digestion. Both wild-type complex and complexes defective in the Msh2p ATPase displayed ATPase activities that were modulated by mismatch and homoduplex DNA substrates. Complexes defective in the Msh6p ATPase, however, displayed weak ATPase activities that were unaffected by the presence of DNA substrate. The results from these studies suggest that the Msh2p and Msh6p subunits of the Msh2p-Msh6p complex play important and coordinated roles in postmismatch recognition steps that involve ATP hydrolysis. Furthermore, our data support a model whereby Msh6p uses its ATP binding or hydrolysis activity to coordinate mismatch binding with additional mismatch repair components.     

Reduced COX-2 protein in colorectal cancer with defective mismatch repair

Most colorectal adenomas and carcinomas arise in the setting of chromosomal instability characterized by progressive loss of heterozygosity. In contrast, approximately 15-20% of colorectal neoplasms arise through a distinct genetic pathway characterized by microsatellite instability (MSI) associated with frequent loss of expression of one of the DNA mismatch repair enzymes, most often hMLH1 or hMSH2. These distinct genetic pathways are reflected by differences in tumor histopathology, distribution in the colon, prognosis, and dwell time required for progression from adenoma to carcinoma. To determine whether these two groups of tumors differ in their expression of cyclooxygenase-2 (COX-2), a putative chemopreventative target, immunostaining for this protein was performed in colorectal cancers categorized by the presence (n = 41) and absence (n = 66) of defective mismatch repair. Defective mismatch repair was defined by the presence of tumor microsatellite instability (MSI-H, > or =40% of markers demonstrating instability) and by the absence of protein expression for either hMLH1 or hMSH2. Overall, our results showed that low or absent COX-2 staining was significantly more common among tumors with defective mismatch repair (P = 0.001). Other features predictive of low COX-2 staining included marked tumor infiltrating lymphocytosis, and solid/cribiform or signet ring histological patterns. These observations indicate that colorectal cancers with molecular and phenotypic characteristics of defective DNA mismatch repair express lower levels of COX-2. The clinical implications of this biological distinction remain unknown but should be considered when assessing the efficacy of COX-2 inhibitors for chemoprevention in patients whose tumors may arise in the setting of defective DNA mismatch repair.     

Mlh1 is unique among mismatch repair proteins in its ability to promote crossing-over during meiosis

In eukaryotes, homologs of the bacterial MutS and MutL proteins function in DNA mismatch repair and recombination pathways. The mutL homolog MLH1 is required for nuclear mismatch repair. Previously, cytological analysis of MLH1-deficient mice has implied a role for Mlh1 in crossing-over during meiosis. Here we demonstrate that Saccharomyces cerevisiae diploids containing a deletion of MLH1 have reduced crossing-over in addition to a deficiency in the repair of mismatched DNA during meiosis. Absence of either of the meiosis-specific mutS homologs Msh4 or Msh5 results in a similar reduction in crossing-over. Analysis of an mlh1 msh4 double mutant suggests that both genes act in the same pathway to promote crossing-over. All genetic markers analyzed in mlh1 mutants display elevated frequencies of non-Mendelian segregation. Most of these events are postmeiotic segregations that represent unrepaired heteroduplex. These data suggest that either restorational repair is frequent or heteroduplex tracts are shorter in wild-type cells. Comparison of mlh1 segregation data with that of pms1, msh2, msh3, and msh6 mutants show that the ability to promote crossing-over is unique to MLH1. Taken together these observations indicate that both crossing-over and gene conversion require MutS and MutL functions and that Mlh1 represents an overlap between these two pathways. Models of Mlh1 function are discussed.     

Mitotic crossovers between diverged sequences are regulated by mismatch repair proteins in Saccaromyces cerevisiae

Mismatch repair systems correct replication- and recombination-associated mispaired bases and influence the stability of simple repeats. These systems thus serve multiple roles in maintaining genetic stability in eukaryotes, and human mismatch repair defects have been associated with hereditary predisposition to cancer. In prokaryotes, mismatch repair systems also have been shown to limit recombination between diverged (homologous) sequences. We have developed a unique intron-based assay system to examine the effects of yeast mismatch repair genes (PMS1, MSH2, and MSH3) on crossovers between homologous sequences. We find that the apparent antirecombination effects of mismatch repair proteins in mitosis are related to the degree of substrate divergence. Defects in mismatch repair can elevate homologous recombination between 91% homologous substrates as much as 100-fold while having only modest effects on recombination between 77% homologous substrates. These observations have implications for genome stability and general mechanisms of recombination in eukaryotes.     

Enhanced intestinal adenomatous polyp formation in Pms2-/-;Min mice

Analysis of two human familial cancer syndromes, hereditary nonpolyposis colorectal cancer and familial adenomatous polyposis, indicates that mutations in either one of four DNA mismatch repair gene homologues or the adenomatous polyposis coli (APC) gene, respectively, are important for the development of colorectal cancer. To further investigate the role of DNA mismatch repair in intestinal tumorigenesis, we generated mice with mutations in both Apc and the DNA mismatch repair gene, Pms2. Whereas Pms2-deficient mice do not develop intestinal tumors, mice deficient in Pms2 and heterozygous for Min, an allele of Apc, develop approximately three times the number of small intestinal adenomas and four times the number of colon adenomas relative to Min and Pms2+/-;Min mice. Although Pms2 deficiency clearly increases adenoma formation in the Min background, histological analysis indicated no clear evidence for progression to carcinoma.     

Evidence for involvement of yeast proliferating cell nuclear antigen in DNA mismatch repair

DNA mismatch repair plays a key role in the maintenance of genetic fidelity. Mutations in the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. The proliferating cell nuclear antigen (PCNA) is essential for DNA replication, where it acts as a processivity factor. Here, we identify a point mutation, pol30-104, in the Saccharomyces cerevisiae POL30 gene encoding PCNA that increases the rate of instability of simple repetitive DNA sequences and raises the rate of spontaneous forward mutation. Epistasis analyses with mutations in mismatch repair genes MSH2, MLH1, and PMS1 suggest that the pol30-104 mutation impairs MSH2/MLH1/PMS1-dependent mismatch repair, consistent with the hypothesis that PCNA functions in mismatch repair. MSH2 functions in mismatch repair with either MSH3 or MSH6, and the MSH2-MSH3 and MSH2-MSH6 heterodimers have a role in the recognition of DNA mismatches. Consistent with the genetic data, we find specific interaction of PCNA with the MSH2-MSH3 heterodimer.     

Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair

The genomes of all eukaryotes contain tracts of DNA in which a single base or a small number of bases is repeated. Expansions of such tracts have been associated with several human disorders including the fragile X syndrome. In addition, simple repeats are unstable in certain forms of colorectal cancer, suggesting a defect in DNA replication or repair. We show here that mutations in any three yeast genes involved in DNA mismatch repair (PMS1, MLH1 and MSH2) lead to 100- to 700-fold increases in tract instability, whereas mutations that eliminate the proof-reading function of DNA polymerases have little effect. The meiotic stability of the tracts is similar to the mitotic stability. These results suggest that tract instability is associated with DNA polymerases slipping during replication, and that some types of colorectal cancer may reflect mutations in genes involved in DNA mismatch repair.     

The carcinogenic response in mice to the topical application of propane sultone to the skin

The carcinogenic effects of limited and repeated skin applications of propane sultone were investigated in three strains of mice, CF1, C3H and CBah (a hairless strain). Propane sultone was shown to be carcinogenic when given as a single application of a 25% w/v solution in toluene and also following twice weekly application of a 2.5% w/v solution for up to 58 weeks. More limited exposure to 2.5% w/v solutions of propane sultone resulted in a few skin tumours, although the incidences were not statistically significant. Most neoplasms were papillomas or carcinomas, although a small number of mesenchymal tumours of dermal origin also developed. No skin neoplasms were found in any control mice. The skin application of propane sultone was associated with a statistically significant increase in the incidence of systematic neoplasia in CFl and C3H mice. The exposed CFl mice had a higher incidence of neoplasms of lymphoreticular and lung origin, while female C3H mice showed a higher incidence of mammary gland and uterine tumours. In mice exposed to beta-propiolactone as a positive control, neoplasms developed at the site of application but, there was no evidence of increased systemic neoplasis in contrast to the findings with ptopane sultone.     

Mismatch repair co-opted by hypermutation

Mice homozygous for a disrupted allele of the mismatch repair gene Pms2 have a mutator phenotype. When this allele is crossed into quasi-monoclonal (QM) mice, which have a very limited B cell repertoire, homozygotes have fewer somatic mutations at the immunoglobulin heavy chain and lambda chain loci than do heterozygotes or wild-type QM mice. That is, mismatch repair seems to contribute to somatic hypermutation rather than stifling it. It is suggested that at immunoglobulin loci in hypermutable B cells, mismatched base pairs are "corrected" according to the newly synthesized DNA strand, thereby fixing incipient mutations instead of eliminating them.     

DNA mismatch repair catalyzed by extracts of mitotic, postmitotic, and senescent Drosophila tissues and involvement of mei-9 gene function for full activity

Extracts of Drosophila embryos and adults have been found to catalyze highly efficient DNA mismatch repair, as well as repair of 1- and 5-bp loops. For mispairs T.G and G.G, repair is nick dependent and is specific for the nicked strand of heteroduplex DNA. In contrast, repair of A.A, C.A, G.A, C.T, T.T, and C.C is not nick dependent, suggesting the presence of glycosylase activities. For nick-dependent repair, the specific activity of embryo extracts was similar to that of extracts derived from the entirely postmitotic cells of young and senescent adults. Thus, DNA mismatch repair activity is expressed in Drosophila cells during both development and aging, suggesting that there may be a function or requirement for mismatch repair throughout the Drosophila life span. Nick-dependent repair was reduced in extracts of animals mutant for the mei-9 gene. mei-9 has been shown to be required in vivo for certain types of DNA mismatch repair, nucleotide excision repair (NER), and meiotic crossing over and is the Drosophila homolog of the yeast NER gene rad1.     

Mismatch repair protein

A DNA mismatch repair system exists that repairs mispaired bases formed during DNA replication and genetic recombination. Genetic defects in this mismatch repair system are known to increase the rate of spontaneous mutation in Escherichia coli. Some cases of inherited cancer are associated with inherited defects of mismatch repair genes, showing the importance of the mismatch repair system in maintenance of genetic stability and avoidance of cancer susceptibility. This review focused on what is known about the mechanisms of mismatch repair in human cells and the relationship between defects in mismatch repair and carcinogenesis.     

Differential induction of c-Jun NH2-terminal kinase and c-Abl kinase in DNA mismatch repair-proficient and -deficient cells exposed to cisplatin

The c-Abl nonreceptor tyrosine kinase and the c-Jun NH2-terminal kinase (JNK/stress-activated protein kinase) are activated during the injury response to the DNA-damaging agent cisplatin. Loss of DNA mismatch repair activity results in resistance to cisplatin in human cancer cells, suggesting that the mismatch repair proteins function as a detector for cisplatin DNA adducts. To identify signaling pathways activated by this detector, we investigated the effect of the loss of DNA mismatch repair function on the ability of cisplatin to activate the JNK and c-Abl kinases. The results demonstrate that cisplatin activates JNK kinase 3.8 +/- 0.2-fold more efficiently in DNA mismatch repair-proficient than repair-deficient cells, and that activation of c-Abl is completely absent in the DNA mismatch repair-deficient cells. Furthermore, the results show that cisplatin-induced activation of JNK occurs through a stress-activated protein kinase/extracellular signal-regulated kinase kinase 1-independent mechanism. We conclude that activation of JNK and c-Abl by cisplatin is in part dependent upon the integrity of DNA mismatch repair function, suggesting that these kinases are part of the signal transduction pathway activated when mismatch repair proteins recognize cisplatin adducts in DNA.     

Human MLH1 deficiency predisposes to hematological malignancy and neurofibromatosis type 1

Heterozygous germ-line mutations in the DNA mismatch repair genes lead to hereditary nonpolyposis colorectal cancer. The disease susceptibility of individuals who constitutionally lack both wild-type alleles is unknown. We have identified three offspring in a hereditary nonpolyposis colorectal cancer family who developed hematological malignancy at a very early age, and at least two of them displayed signs of neurofibromatosis type 1 (NF1). DNA sequence analysis and allele-specific amplification in two siblings revealed a homozygous MLH1 mutation (C676T-->Arg226Stop). Thus, a homozygous germ-line MLH1 mutation and consequent mismatch repair deficiency results in a mutator phenotype characterized by leukemia and/or lymphoma associated with neurofibromatosis type 1.     

DHFR/MSH3 amplification in methotrexate-resistant cells alters the hMutSalpha/hMutSbeta ratio and reduces the efficiency of base-base mismatch repair

The level and fate of hMSH3 (human MutS homolog 3) were examined in the promyelocytic leukemia cell line HL-60 and its methotrexate-resistant derivative HL-60R, which is drug resistant by virtue of an amplification event that spans the dihydrofolate reductase (DHFR) and MSH3 genes. Nuclear extracts from HL-60 and HL-60R cells were subjected to an identical, rapid purification protocol that efficiently captures heterodimeric hMutSalpha (hMSH2. hMSH6) and hMutSbeta (hMSH2.hMSH3). In HL-60 extracts the hMutSalpha to hMutSbeta ratio is roughly 6:1, whereas in methotrexate-resistant HL-60R cells the ratio is less than 1:100, due to overproduction of hMSH3 and heterodimer formation of this protein with virtually all the nuclear hMSH2. This shift is associated with marked reduction in the efficiency of base-base mismatch and hypermutability at the hypoxanthine phosphoribosyltransferase (HPRT) locus. Purified hMutSalpha and hMutSbeta display partial overlap in mismatch repair specificity: both participate in repair of a dinucleotide insertion-deletion heterology, but only hMutSalpha restores base-base mismatch repair to extracts of HL-60R cells or hMSH2-deficient LoVo colorectal tumor cells.     

Fasting prevents experimental murine colitis produced by dextran sulfate sodium and decreases interleukin-1 beta and insulin-like growth factor I messenger ribonucleic acid

Cytokines and insulin-like growth factors (IGFs) are involved in the induction and/or perpetuation of inflammatory bowel disease. The effect of fasting on inflammatory bowel disease was studied in a mouse experimental model of acute colitis caused by adding dextran sulfate sodium (DSS) to drinking water. Animals were either fed ad libitum or fasted (water only) for 2 days before death. Inflammation and tissue damage, measured as a colitis activity score, were markedly reduced in fasted (2.4 +/- 0.1) compared to fed (5.3 +/- 0.1) DSS animals (P < 0.0001). Colon interleukin-1 beta (IL-1 beta), IGF-I, and tumor necrosis factor-alpha messenger RNAs (mRNAs) were quantified by Northern blot hybridization and expressed as a percentage of mRNA abundance in fed controls. In DSS mice, IL-1 beta mRNA was elevated in the fed group (954 +/- 155%; P < 0.001), but was suppressed in fasted animals (71.1 +/- 11%). IGF-I mRNA also was elevated in fed DSS mice (421 +/- 71%; P < 0.01). This increase was attenuated in fasted DSS mice (202 +/- 17%; P < 0.01 compared to fed DSS mice). Tumor necrosis factor-alpha mRNA was increased in fed DSS mice (162 +/- 15%; P < 0.01), but was not significantly lower in fasted animals. By in situ hybridization, IL-1 beta mRNA was localized to the lamina propria of colonic mucosa in fed DSS animals, but was not detectable in other groups. We conclude that fasting has a protective effect on the progression of acute DSS, induced colitis. This is associated with decreased expression of IL-1 beta and IGF-I mRNAs in the colon.