Genome instability and DNA damage accumulation in gene-targeted mice

Six major pathways for DNA repair have been identified. These include (1) DNA repair by direct reversal, (2) base excision repair, (3) mismatch repair, (4) nucleotide excision repair, (5) homologous recombination, and (6) non-homologous end-joining. In addition, several other cellular processes influence the response to DNA damage. The generation of gene-targeted organisms is crucial for assessing the relative contribution of single DNA repair proteins and DNA repair pathways in maintaining genome stability. In particular, the accumulation of DNA damage, mutations and cancer in unexposed gene-targeted animals illuminates the spontaneous load of a particular lesion and the relative significance of a single gene in a specific pathway. Strategies for the generation of gene-targeted mice have been available for 15 years and more than 100 different genes relevant to DNA repair have been targeted. This review describes some important progress made toward understanding spontaneous DNA damage and its repair, exemplified through one, or a few, gene-targeted mice from each major DNA repair pathway.     

Using mouse models to investigate the biological and physiological consequences of defects in the Fanconi anaemia/breast cancer DNA repair signalling pathway

Fanconi anaemia (FA) is a rare, predominantly autosomal recessive syndrome (with one X-linked form) that results in congenital defects, abnormal haematopoiesis and a greatly increased risk of solid tumours in humans. Mutations in at least 14 different genes have been shown to cause FA, and several of these genes, including FANCJ/BRIP1, FANCD1/BRCA2 and FANCN/PALB2, also predispose to breast cancer in heterozygote carriers. The FA genes code for proteins that act in complexes to coordinate the repair of damaged DNA, and thus the FA repair network is intimately linked with hereditary breast cancer. Much remains to be learnt about the functions and interactions of the FA proteins and one experimental approach involves the generation of mice that are deficient in various FA genes. Mouse models for FANCN/PALB2 have recently been generated, including one reported in a recent issue of The Journal of Pathology. Given the pivotal role of the PALB2 protein, which interacts with both BRCA1 and BRCA2, these mice provide valuable insights into the FA phenotype and mechanisms of tumourigenesis caused by disruption of the FA protein network.     

Multiple loci are linked with anti-red blood cell antibody production in NZB mice -- comparison with other phenotypes implies complex modes of action

The New Zealand Black (NZB) mouse strain is a model of autoimmune haemolytic anaemia (AHA) and systemic lupus erythematosus (SLE), characterized by the production of anti-red blood cell (RBC) antibodies and anti-nuclear antibodies (ANA), respectively. A linkage analysis was carried out in an (NZB x BALB/c) F(2) cross in order to identify loci involved in the production of both anti-RBC IgM and IgG antibodies. These regions of linkage were compared with linkage data to ANA from the same cohort and other linkage analyses involving New Zealand mice. Four previously described NZB loci linked to anti-RBC antibodies were confirmed, and eight novel loci linked to this trait were also mapped: five of which were of NZB origin, and three derived from the non-autoimmune BALB/c background. A comparison between loci linked with anti-RBC antibodies and ANA demonstrated many that co-localize, suggesting the presence of genes that result in the general breaking of tolerance to self-antigen. Furthermore, the observation that some loci were associated only with the anti-RBC response suggests an antigen specific mechanism in addition to a general breaking of tolerance. A locus linked with anti-RBC antibodies and ANA on distal chromosome 7 in this cohort is orthologous to one on the q arm of human chromosome 11, a region linked to AHA and ANA in human SLE.     

SJLB mice develop tauopathy-induced parkinsonism

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is an inherited dementia caused by tauopathy. Recently, we established the N279K mutant human tau transgenic mice SJLB. Although SJLB mice show cognitive dysfunction with insoluble tau in the brain, it has remained unclear whether they show signs of parkinsonism. To clarify this issue, we studied whether SJLB mice in fact develop parkinsonism. Behavioral analysis showed shorter stride length than that of non-transgenic control mice in the footprint test and movement disorder in the pole test, thus mimicking some features of human parkinsonism. We also found that these symptoms were not affected by dopamine treatment. These results indicate that SJLB mice show signs of parkinsonism and they could be of usefulness not only for studies of dementing disease but also of parkinsonism induced by tauopathy.     

Autophagy-deficient mice develop multiple liver tumors

Autophagy is a major pathway for degradation of cytoplasmic proteins and organelles, and has been implicated in tumor suppression. Here, we report that mice with systemic mosaic deletion of Atg5 and liver-specific Atg7^−/− mice develop benign liver adenomas. These tumor cells originate autophagy-deficient hepatocytes and show mitochondrial swelling, p62 accumulation, and oxidative stress and genomic damage responses. The size of the Atg7^−/− liver tumors is reduced by simultaneous deletion of p62. These results suggest that autophagy is important for the suppression of spontaneous tumorigenesis through a cell-intrinsic mechanism, particularly in the liver, and that p62 accumulation contributes to tumor progression.     

小鼠骨损伤模型在骨修复机制研究中的应用

骨折是常见多发病,建立标准的骨损伤动物模型是进行骨折实验研究的基础,由于小鼠具有和人类相似的遗传背景,并且可进行多种遗传修饰等优势,因此在骨折研究中的应用越来越广泛。     

FANCD2 protein is expressed in proliferating cells of human tissues that are cancer-prone in Fanconi anaemia

Fanconi anaemia (FA) is an inherited form of progressive pancytopenia associated with developmental defects, chromosomal instability, and cancer predisposition. At least seven distinct FA proteins function in concert to protect the genome, a key step being the activation of FANCD2 by mono-ubiquitination. This paper reports an immunohistochemical analysis of FANCD2 expression in normal human tissue. The highest expression was observed in maturing spermatocytes and fetal oocytes (consistent with a role for FANCD2 in meiosis) and in germinal centre cells of the spleen, tonsil, and lymph nodes (consistent with a role in proliferation). FANCD2 expression was also seen in tissues predisposed to cancer development in FA patients: haematopoietic cells, especially in the fetus, and squamous cell epithelia, particularly in the head and neck region and uterine cervix. FANCD2 expression was also occasionally seen in the breast and Fallopian tube epithelium, the respiratory epithelium of the trachea, and the exocrine cells of the pancreas, indicating that these tissues may also be cancer-prone in FA. FANCD2 expression is frequently expressed in proliferating cells as demonstrated by Ki-67 immunofluorescence double staining, consistent with a function of FANCD2 in DNA replication.     

Lack of chloroform-induced DNA repair in vitro and in vivo in hepatocytes of female B6C3F1 mice

Chloroform has been shown to induce hepatocellular carcinomas in female B6C3F₁ mice when administered by gavage, but not when given in drinking water. When administered in corn oil at the carcinogenic doses of 238 and 477 mg/kg, chloroform induced necrosis and sustained regenerative cell proliferation in the liver. To investigate the mode of action of tumor induction in the target cells, the ability of chloroform to induce unscheduled DNA synthesis (UDS) was examined in the in vitro and in vivo hepatocyte DNA repair assays. In the in vitro assay, primary hepatocyte cultures from female B6C3F₁ mice were incubated with concentrations from 0.01 to 10 mM chloroform in the presence of ³H-thymidine. UDS was assessed by quantitative autoradiography. No induction of DNA repair was observed at any concentration. In the in vivo assay, animals were treated by gavage with 238 and 477 mg/kg chloroform in corn oil. Primary hepatocyte cultures were prepared 2 and 12 hr later, incubated with ³H-thymidine, and assessed for induction of UDS as above. No DNA repair activity was seen at either dose or at either timepoint. These negative results in the target organ are consistent with the concept that neither chloroform nor its metabolites are directly DNA reactive and that the carcinogenicity of chloroform is secondary to induced cytolethality and regenerative cell proliferation. © 1994 Wiley-Liss, Inc.     

Regulation of DNA cross-link repair by the Fanconi anemia/BRCA pathway

The maintenance of genome stability is critical for survival, and its failure is often associated with tumorigenesis. The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links (ICLs), and a germline defect in the pathway results in FA, a cancer predisposition syndrome driven by genome instability. Central to this pathway is the monoubiquitination of FANCD2, which coordinates multiple DNA repair activities required for the resolution of ICLs. Recent studies have demonstrated how the FA pathway coordinates three critical DNA repair processes, including nucleolytic incision, translesion DNA synthesis (TLS), and homologous recombination (HR). Here, we review recent advances in our understanding of the downstream ICL repair steps initiated by ubiquitin-mediated FA pathway activation.     

Duchenne's muscular dystrophy: animal models used to investigate pathogenesis and develop therapeutic strategies

Duchenne's muscular dystrophy (DMD) is a lethal childhood disease caused by mutations of the dystrophin gene, the protein product of which, dystrophin, has a vital role in maintaining muscle structure and function. Homologues of DMD have been identified in several animals including dogs, cats, mice, fish and invertebrates. The most notable of these are the extensively studied mdx mouse, a genetic and biochemical model of the human disease, and the muscular dystrophic Golden Retriever dog, which is the nearest pathological counterpart of DMD. These models have been used to explore potential therapeutic approaches along a number of avenues including gene replacement and cell transplantation strategies. High-throughput screening of pharmacological and genetic therapies could potentially be carried out in recently available smaller models such as zebrafish and Caenorhabditis elegans. It is possible that a successful treatment will eventually be identified through the integration of studies in multiple species differentially suited to addressing particular questions.     

FANCJ suppresses microsatellite instability and lymphomagenesis independent of the Fanconi anemia pathway

Microsatellites are short tandem repeat sequences that are highly prone to expansion/contraction due to their propensity to form non-B-form DNA structures, which hinder DNA polymerases and provoke template slippage. Although error correction by mismatch repair plays a key role in preventing microsatellite instability (MSI), which is a hallmark of Lynch syndrome, activities must also exist that unwind secondary structures to facilitate replication fidelity. Here, we report that Fancj helicase-deficient mice, while phenotypically resembling Fanconi anemia (FA), are also hypersensitive to replication inhibitors and predisposed to lymphoma. Whereas metabolism of G4-DNA structures is largely unaffected in Fancj^−/− mice, high levels of spontaneous MSI occur, which is exacerbated by replication inhibition. In contrast, MSI is not observed in Fancd2^−/− mice but is prevalent in human FA-J patients. Together, these data implicate FANCJ as a key factor required to counteract MSI, which is functionally distinct from its role in the FA pathway.     

Senescent BALB/c mice are able to develop resistance to Leishmania major infection

Aging has been associated with a decline in immunocompetence and resistance to infections, partially due to dysregulated NO production by macrophages and deficits in mounting Th2 cell responses. We wondered if these alterations would reverse the immune response in experimental leishmaniasis. Bone-marrow-derived macrophages from 2- and 18-month-old (senescent) C57BL/6 or BALB/c mice showed no marked difference in leishmanicidal functions. In vivo infections of resistant C57BL/6 mice with Leishmania major revealed no difference between senescent and young mice. However, among susceptible BALB/c mice, senescent animals showed less foot-pad swelling than young mice, and 40 to 60% of them even showed healing of ulcers, reduced parasite dissemination, and a Th1 cell response. These changes were associated with a spontaneous release of interleukin-12 (IL-12) by macrophages from aged but not from young mice. Since exogenous microbial stimulation can influence immune responses during aging, we also infected senescent mice who were raised under specific-pathogen-free (SPF) conditions. They showed neither resistance nor a Th1 response, but their macrophages still spontaneously released IL-12. A microbiological analysis showed that conventionally kept mice, but not SPF mice, had experienced infection with murine hepatitis virus (MHV), an infection associated with a Th1-like response. We conclude that for the reversal of the immune response, senescence is the premier requirement but needs to be completed by another mandatory event such as microbial stimulation. One of the age-related, but not environment-related, factors is the spontaneous release of IL-12 by macrophages, while confrontation with MHV presents an environment-related difference, with both having the potential to support a Th1 response.     

Behavioural mechanisms affecting energy regulation in mice prone or resistant to diet- induced obesity

We investigated inbred SWR/J and AKR/J mice, two established models for different susceptibility to diet-induced obesity (DIO), to scrutinize the contribution of physical activity and energy assimilation to the etiology of developing obesity. Body mass gain and body composition of mice fed a high-energy (HE) or a low caloric control diet were monitored. In parallel, assimilated energy, locomotor activity and thermoregulatory behaviour were measured. Activity was continuously registered by radio telemetry and, in addition, Open Field (OF) behaviour was used as a quick screening tool for spontaneous activity before and after the feeding trial. Energy assimilation was increased in both strains on HE (AKR/J: + 60.7% and SWR/J: + 42.8%) but only in AKR/J, body mass (+ 8.1%) and fat mass (+ 40.7%) were significantly elevated. As a trend, total home cage activity was increased and was more scattered in SWR/J. Interestingly, HE stimulated OF activity only in SWR/J in the second trial at the end of the feeding experiment. The spatial pattern of OF activity also differed between strains with obese mice avoiding the core area. Under housing conditions, nest building behaviour was more pronounced in AKR/J. To further evaluate OF behaviour as a marker for spontaneous activity an obese mouse line was investigated. Mice lacking the leptin receptor (db/db) showed already before the onset of obesity lowest activity levels in OF.Adjustment of energy intake, higher activity levels and energy consuming thermoregulatory behaviour are mechanisms employed by SWR/J mice to dissipate excess energy as a defence against the onset of obesity. Therefore our results deciphering mechanisms of DIO-sensitivity in mice contribute to the understanding of inter-individual differences in body weight development in an adipogenic environment.     

FANCL gene mutations in premature ovarian insufficiency

The Fanconi anemia (FA) pathway is mainly involved in DNA interstrand crosslinks (ICLs) repair in the genome. Several FA genes, including FANCD1/BRCA2, FANCM, and FANCU/XRCC2, have been identified as causative genes for premature ovary insufficiency (POI). Fanconi anemia group L protein (FANCL) cooperates with FANCT/UBE2T to ubiquitinate the FANCI‐D2 dimer, which is a crucial event in the process of ICLs repair. Fancl‐knockout mice phenocopy human POI, but the role of FANCL mutations in POI pathogenesis has not been confirmed. In the present work, potentially pathogenic mutations in the FANCL gene were screened in 200 Chinese patients with idiopathic POI and in 200 matched controls. Two novel heterozygous frameshift mutations, c.1048_1051delGTCT (p.Gln350Valfs*18) and c.739dupA (p.Met247Asnfs*4), were identified in the FANCL gene in POI patients but not in controls. Wild‐type FANCL protein was predominantly localized in the nuclei, while both mutant FANCL proteins were retained in the cytoplasm. In addition, the FANCL variants exhibited impaired ubiquitin‐ligase activity and compromised DNA repair ability after mitomycin C treatment. Furthermore, the FANCL variants were deleterious and might be associated with haploinsufficiency. Our results show that FANCL mutations are potentially causative for POI by disrupting DNA damage repair processes.     

Diverse antigen specificity of erythrocyte-reactive monoclonal autoantibodies from NZB mice

The specificities of a panel of erythrocyte-reactive MoAbs derived from NZB mice with autoimmune haemolytic anaemia (AIHA) were determined by immunoprecipitation and immunoblotting. Of the eight antibodies, two (IgG1 MoAb 105-2H and IgG2a MoAb 34-3C) immunoprecipitated a 105-kD component identified as the erythrocyte anion channel band 3. A similar band was also immunoprecipitated by the IgG2b MoAb 34-2B when used at relatively high concentrations, but none of the remaining hybridoma antibodies precipitated any labelled erythrocyte components. In immunoblotting experiments only 34-2B reacted with band 3, indicating that the epitope recognized by this MoAb is robust and differs from the determinant(s) recognized by 105-2H and 34-3C. The remaining MoAbs to react by immunoblotting were the IgM antibodies IE10 and 4C8, both of which bound to a doublet corresponding to band 4.1 from the internal erythrocyte membrane skeleton. Of the three MoAbs which gave negative results in immunoprecipitation and immunoblotting, the IgM antibodies 103-7E and 106-10E reacted poorly with intact erythrocytes by flow cytometry, but the IgG1 antibody 31-9D bound well. ELISAs demonstrated that all four IgM MoAbs are polyreactive, since they bound to histones from a panel of nuclear antigens, and additionally 103-7E reacted with phosphatidyl choline. It is concluded that band 3 is an important autoantigen in NZB AIHA. However, since 3/5 haemolytic MoAbs failed to precipitate this antigen, either these antibodies represent minor components of the total autoantibody response, or responses to diverse possibly non-protein surface antigens also contribute to the pathogenesis of the disease.     

Proliferative potential and DNA repair in lymphocytes from short-lived and long-lived strains of mice, relation to aging

The replicative capacity and DNA repair ability of spleen lymphocytes of young and old mice from short-lived and long-lived strains were studied. DNA repair after both UV- and gamma-induced damage was investigated. Proliferation after Con A decreased as a function of age in both mouse strains and paralleled an age-associated decline in repair of DNA damage induced by either UV or gamma-irradiation. Compared to the long-lived, the short-lived strain displayed an earlier impairment of both proliferative and repair potentials. DNA repair after gamma-induced damage only occurred if lymphocytes were stimulated to proliferate. Resting lymphocytes appeared unable to repair strand breaks. By contrast, DNA repair of UV-induced damage showed two components: one was dependent on the cell proliferative state, the other was not. Both components were stimulated or induced by mitogen. Resting lymphocytes were able to perform an appreciable amount of repair after UV irradiation. Our results suggest that resting or post-mitotic cells possess a greater possibility to regulate repair of UV-induced than gamma-induced damage. We speculate that it may be the level of this proliferation-independent, but mitogen inducible form of repair which correlates with maximum life-spans between species, thereby explaining why repair of UV- but not gamma-induced damage reveals such a correlation.     

L-Selectin-deficient SJL and C57BL/6 mice are not resistant to experimental autoimmune encephalomyelitis

L-selectin has been suggested to play a role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Here we demonstrate that L-selectin(-/-) SJL mice are susceptible to proteolipid protein (PLP)-induced EAE because the compromised antigen-specific T cell proliferation in peripheral lymph nodes is fully compensated by the T cell response raised in their spleen. Transfer of PLP-specific T cells into syngeneic recipients induced EAE independent of the presence or absence of L-selectin on PLP-specific T cells or in the recipient. Leukocyte infiltration into the central nervous system parenchyma was detectable independent of the mode of disease induction and the presence or absence of L-selectin. In addition, we found L-selectin(-/-) C57BL/6 mice to be susceptible to myelin oligodendrocyte glycoprotein-induced EAE. Taken together, we demonstrate that in SJL and C57BL/6 mice L-selectin is not required for EAE pathogenesis. The apparent discrepancy of our present observation to previous findings, demonstrating a role of L-selectin in EAE pathogenesis in C57BL/6 mice or myelin-basic protein (MBP)-specific TCR-transgenic B10.PL mice, may be attributed to background genes rather than L-selectin and to a unique role of L-selectin in EAE pathogenesis in MBP-TCR-transgenic mice.     

Mouse models of DNA polymerases

In 1956, Arthur Kornberg discovered the mechanism of the biological synthesis of DNA and was awarded the Nobel Prize in Physiology or Medicine in 1959 for this contribution, which included the isolation and characterization of Escherichia coli DNA polymerase I. Now there are 15 known DNA polymerases in mammalian cells that belong to four different families. These DNA polymerases function in many different cellular processes including DNA replication, DNA repair, and damage tolerance. Several biochemical and cell biological studies have provoked a further investigation of DNA polymerase function using mouse models in which polymerase genes have been altered using gene‐targeting techniques. The phenotypes of mice harboring mutant alleles reveal the prominent role of DNA polymerases in embryogenesis, prevention of premature aging, and cancer suppression.