Genetic model of some human hereditary diseases: features of behavior, neurochemistry and morphology of the brain in mice of the 101/HY line

Studies of behavior, neurophysiological reactions, neuromediator synthesis and brain structure of mice of the 101/HY strain (including those of the authors) are reviewed. This mouse strain is characterized by a chromosomal instability because of a recessive mutation mutator-1 (mut-1) and the defective DNA excision repair. Experimental studies revealed a number of behavioral and neurological deviations in the 101/HY as compared to the CBA and the C3H strains. These are abnormalities in spatial orientation, altered fear and anxiety reactions, anomalous locomotion, seizure developing in response to agents of various nature, and disturbances of the central nervous system, both structural and biochemical. Genome instability results in a number of neurological mutations, that may lead to the phenotypical effects observed in the 101/HY mice. Since the 101/HY mice partially display signs of severe human hereditary diseases caused by chromosomal instability and defective DNA repair, they can serve as a promising genetic model for these and other diseases related to impairment of the central nervous system.     

Replication delay along FRA7H, a common fragile site on human chromosome 7, leads to chromosomal instability

Common fragile sites are specific chromosomal loci that show gaps, breaks, or rearrangements in metaphase chromosomes under conditions that interfere with DNA replication. The mechanism underlying the chromosomal instability at fragile sites was hypothesized to associate with late replication time. Here, we aimed to investigate the replication pattern of the common fragile site FRA7H, encompassing 160 kb on the long arm of human chromosome 7. Using in situ hybridization on interphase nuclei, we revealed that the replication of this region is initiated relatively early, before 30% of S phase is completed. However, a high fraction ( approximately 35%) of S-phase nuclei showed allelic asynchrony, indicating that the replication of FRA7H is accomplished at different times in S phase. This allelic asynchrony is not the result of a specific replication time of each FRA7H allele. Analysis of the replication pattern of adjacent clones along FRA7H by using cell population and two-color fluorescent in situ hybridization analyses showed significant differences in the replication of adjacent clones, under normal growth condition and upon aphidicolin treatment. This pattern significantly differed from that of two nonfragile regions which showed a coordinated replication under both conditions. These results indicate that aphidicolin is enhancing an already existing difference in the replication time along the FRA7H region. Based on our replication analysis of FRA7H and on previous analysis of the common fragile site FRA3B, we suggest that delayed replication is underlying the fragility at aphidicolin-induced common fragile sites.     

An unusual type of locomotion in mice of line 101/Hy]

An unusual type of locomotion--backward movements--was revealed in the 101/HY mouse strain characterized by a chromosomal instability and the defect in DNA repair. The backward movements were found in 60% of mice belonging to the population under study. The manifestation of the character was found to vary: it was high in 16% of the animals. No age and sex differences were found. The backward movements in the 101/HY mouse strain were somewhat similar to the peculiarity in locomotion of the neurological hot-foot mouse mutants. The latter had mild anatomical alterations in the cerebellum. No obvious brain pathology was revealed in the 101/HY mice.     

Unscheduled overexpression of human WAPL promotes chromosomal instability

Previously, we have isolated and characterized a novel human gene termed human WAPL that has the characteristics of an oncogene in uterine cervical cancer. WAPL is inducible by human papillomavirus (HPV) E6 and E7 oncoproteins. On the other hand, recent studies have revealed that WAPL regulates sister chromatid resolution by controlling the association of cohesin and chromatin. However, the effects of WAPL overexpression on cervical carcinogenesis are still unclear. Here, we show that WAPL overexpression induces generation of multinucleated cells. In addition, WAPL-overexpressing cells demonstrated increases in chromatid breaks in comparison with control cells. These results were obtained even in HPV-negative cell lines. High frequent premature sister separation by disregulation of cohesin may lead to these results. Thus, our study suggests that unscheduled overexpression of WAPL disturbs mitosis and cytokinesis, and contributes to tumor progression by induction of chromosomal instability (CIN).     

Genomic architecture of human chromosomal diseases

The genomic architecture predisposed to the emergence of DNA copy number variation causing a new class of human chromosomal diseases—reciprocal microdeletion and microduplication syndromes— is reviewed in the paper. The molecular mechanisms of such chromosomal abnormalities are described. The problems of the interpretation of their clinical significance and genotype-phenotype correlations are discussed. The classification of phenotypes due to reciprocal chromosomal microdeletions and microduplications is shown. Published by 2015, reciprocal mutations associated with inherited and congenital human pathology and involving 58 chromosomal regions are summarized.     

Telomere instability in a human cancer cell line.

Telomere maintenance is essential in immortal cancer cells to compensate for DNA lost from the ends of chromosomes, to prevent chromosome fusion, and to facilitate chromosome segregation. However, the high rate of fusion of chromosomes near telomeres, termed telomere association, in many cancer cell lines has led to the proposal that some cancer cells may not efficiently perform telomere maintenance. Deficient telomere maintenance could play an important role in cancer because telomere associations and nondisjunction have been demonstrated to be mechanisms for genomic instability. To investigate this possibility, we have analyzed the telomeres of the human squamous cell carcinoma cell line SQ-9G, which has telomere associations in approximately 75% of the cells in the population. The absence of detectable telomeric repeat sequences at the sites of these telomere associations suggests that they result from telomere loss. The analysis of telomere length by quantitative in situ hybridization demonstrated that, compared to the human squamous cell carcinoma cell line SCC-61 which has few telomere associations, SQ-9G has more extensive heterogeneity in telomere length and more telomeres without detectable telomeric repeat sequences. The dynamics of the changes in telomere length also demonstrated a higher rate of fluctuation in telomere length, both on individual telomeres and coordinately on all telomeres. These results demonstrate that telomere maintenance can play a role in the genomic instability seen in cancer cells.     

Saccharomyces cerevisiae as a model system to define the chromosomal instability phenotype

Translocations, deletions, and chromosome fusions are frequent events seen in cancers with genome instability. Here we analyzed 358 genome rearrangements generated in Saccharomyces cerevisiae selected by the loss of the nonessential terminal segment of chromosome V. The rearrangements appeared to be generated by both nonhomologous end joining and homologous recombination and targeted all chromosomes. Fifteen percent of the rearrangements occurred independently more than once. High levels of specific classes of rearrangements were isolated from strains with specific mutations: translocations to Ty elements were increased in telomerase-defective mutants, potential dicentric translocations and dicentric isochromosomes were associated with cell cycle checkpoint defects, chromosome fusions were frequent in strains with both telomerase and cell cycle checkpoint defects, and translocations to homolog genes were seen in strains with defects allowing homoeologous recombination. An analysis of human cancer-associated rearrangements revealed parallels to the effects that strain genotypes have on classes of rearrangement in S. cerevisiae.     

Two quantitative trait loci affecting progressive hearing loss in 101/H mice

Although recent progress in identifying genes involved in deafness has been remarkable, the genetic basis of progressive hearing loss (or age-related hearing loss) is poorly understood because of the extreme difficulty in studying such a late-onset, complex disease in human populations. Several inbred strains of mice such as 129P1/ReJ, C57BL/6J, DBA/2J, and BALB/cByJ have been reported to exhibit age-related hearing loss and provide valuable models for human nonsyndromic progressive deafness. In this article we show that 101/H mice also exhibit progressive deafness with early onset. Linkage analysis of F₂ populations derived from crosses between the 101/H and the MAI/Pas and MBT/Pas wild-derived mice suggested at least two major quantitative trait loci (QTLs) that influence progressive hearing loss. A first QTL, designated Phl1, was mapped with a maximum LOD score of 6.7 to the centromeric region of Chromosome 17, where no deafness-related QTL has been mapped so far. A second QTL, designated Phl2, mapped to Chromosome 10 and exhibited a maximum LOD score of 5.3. The map position of Phl2 near the well-known QTL of age-related hearing loss (Ahl) suggested the possibility of allelism, although the Ahl mutation itself did not segregate in these crosses. Finally, we found some evidence of epistatic interaction between Phl1 and Phl2. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Tomoji Mashimo, Alexandra E. Erven, and Sarah L. Spiden contributed equally to this work.     

Detection of chromosomal instability in alpha-irradiated and bystander human fibroblasts

There is increasing evidence biological responses to ionizing radiation are not confined to those cells that are directly hit, but may be seen in the progeny at subsequent generations (genomic instability) and in non-irradiated neighbors of irradiated cells (bystander effects). These so called non-targeted phenomena would have significant contributions to radiation-induced carcinogenesis, especially at low doses where only a limited number of cells in a population are directed hit. Here we present data using a co-culturing protocol examining chromosomal instability in alpha-irradiated and bystander human fibroblasts BJ1-htert. At the first cell division following exposure to 0.1 and 1Gy alpha-particles, irradiated populations demonstrated a dose dependent increase in chromosome-type aberrations. At this time bystander BJ1-htert populations demonstrated elevated chromatid-type aberrations when compared to controls. Irradiated and bystander populations were also analyzed for chromosomal aberrations as a function of time post-irradiation. When considered over 25 doublings, all irradiated and bystander populations had significantly higher frequencies of chromatid aberrations when compared to controls (2-3-fold over controls) and were not dependent on dose. The results presented here support the link between the radiation-induced phenomena of genomic instability and the bystander effect.     

Effect of thiophosphamide on the early period of gametogenesis in CBA, 101/H and AKR mice]

On the 12th day of pregnancy CBA, 101/H and AKR mice were given thiophosphamide in a dose of 5 mg/kg bw, and were sacrificed on the 19th day of pregnancy. The action of thiophosphamide on embryonal ovaries and testes was studied by assaying for the population of oocytes and relative number of the different stages of meiotic prophase I; index of the degeneration germ cells; for the population of prospermatogonies and their degeneration; morphometric study of nucleus of nucleolus of prospermatogonies. A significant decrease of germ cells was found in male and female embryos on the 19th day of pregnancy after thiophosphamide injection. Interspecific differences were found as regards the number of germ cells and their proportion in health as well as in response to a single antenatal injection of thiophosphamide.     

Reproducible chromosomal instability of an established Chinese hamster cell line detectable by flow cytometry

The karyotypes of individual cells in clones of the established Chinese hamster cell line display a highly heterogeneous pattern. Unlike situation in individual cells, the flow karyotypes of cloned cell populations are very similar. A comparison of these facts suggests that mostly the same certain chromosomal reorganizations, appearing frequently enough, may occur in the cells. As a result, the whole set of possible variants of reorganized chromosomes appear during few cell cycles, regardless of the initial cell karyotype. This hypothesis is supported by our flow cytometry data. The same small peaks corresponding to rarely met (less than 1 per cell) rear ranged chromosomes appear on flow karyotype histograms of parental cell clones and their secondary subclones. Chromosomes with random gamma or UV irradiation-induced reorganizations do not remain in the cell population, unlike certain reorganization of regular nature.     

Telomere instability in a human tumor cell line expressing a dominant-negative WRN protein

Werner Syndrome (WS) is an autosomal recessive disease characterized by premature aging and chromosome instability. The protein involved in WS, WRN, is a RecQ-type helicase that also has exonuclease activity. WRN has been demonstrated to bind to a variety of other proteins, including RPA, DNA-PKcs, and TRF2, suggesting that WRN is involved in DNA replication, repair, recombination, and telomere maintenance. In culture, WS cells show premature senescence, which can be overcome by transfection with an expression vector containing the gene for the catalytic subunit of telomerase. However, telomerase expression does not eliminate chromosome instability in WS cells, which led to the proposal that telomere loss is not the cause of the high rate of chromosome rearrangements in WS cells. In the present study, we have investigated how a WRN protein containing a dominant-negative mutation (K577M-WRN) influences the stability of telomeres in a human tumor cell line expressing telomerase. The results demonstrate an increased rate of telomere loss and chromosome fusion in cells expressing K577M-WRN. Expression of K577M-WRN results in reduced levels of telomerase activity, however, the absence of detectable changes in average telomere length demonstrates that WRN-associated telomere loss results from stochastic events involving complete telomere loss or loss of telomere capping function. Thus, telomere loss can contribute to chromosome instability in cells deficient in WRN regardless of the expression of telomerase activity.     

Restoration of telomerase activity rescues chromosomal instability and premature aging in Terc-/- mice with short telomeres

Reconstitution of telomerase activity is proposed as a potential gene therapy to prevent, or rescue, age-related diseases produced by critical telomere shortening. However, it is not known whether or not short telomeres are irreversibly damaged. We addressed this by re-introducing telomerase in late generation telomerase-deficient mice, Terc^–/–, which have short telomeres and show severe proliferative defects. For this, we have crossed these mice with Terc^+/– mice and analyzed telomere length, chromosomal instability and premature aging of the progeny. The Terc^–/– progeny had one set of chromosomes with normal telomeres, whereas the other set remained with critically short telomeres; these mice presented chromosomal instability and premature aging. In contrast, Terc^+/– progeny showed all chromosomes with detectable telomeres, and did not show chromosomal instability or premature aging. These results prove that critically short telomeres can be rescued by telomerase, and become fully functional, thus rescuing premature aging. This has important implications for the future design of telomerase-based gene therapy of age-related diseases.     

Polyploid mitosis and depolyploidization promote chromosomal instability and tumor progression in a Notch-induced tumor model

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The radiosensitivity of spermatogonial stem cells in C3H/101 F1 hybrid mice

The radiosensitivity of spermatogonial stem cells of C3H/HeH × 101/H F₁ hybrid mice was determined by counting undifferentiated spermatogonia at 10 days after X-irradiation. During the spermatogenic cycle, differences in radiosensitivity were found, which were correlated with the proliferative activity of the spermatogonial stem cells. In stage VIII_irr, during quiescence, the spermatogonial stem cells were most radiosensitive with a D₀ of 1.4 Gy. In stages XI_irr−V_irr, when the cells were proliferatively active, the D₀ was about 2.6 Gy. Based on the D₀ values for sensitive and resistant spermatogonia and on the D₀ for the total population, a ratio of 45:55% of sensitive to resistant spermatogonial stem cells was estimated for cell killing.

When the present data were compared with data on translocation induction obtained in mice of the same genotype, a close fit was obtained when the translocation yield (Y; in % abnormal cells) after a radiation dose D was described by Y = e^τD, with τ = 1 for the sensitive and τ = 0.1 for the resistant spermatogonial stem cells, with a maximal e^τD of 100.     

Checkpoint failure and chromosomal instability without lymphomagenesis in Mre11(ATLD1/ATLD1) mice

In this study, mice expressing one of the two Mre11 alleles inherited in the human ataxia-telangiectasia like disorder (A-TLD) were derived. The mutation had a profound maternal effect on embryonic viability, revealing an acute requirement for Mre11 complex function in early embryogenesis. Mre11(ATLD1/ATLD1) mice exhibited several indices of impaired ATM function. The mice also exhibited pronounced chromosomal instability. Despite this phenotypic spectrum, the animals were not prone to malignancy. These data indicate that defective cell cycle checkpoints and chromosomal instability are insufficient to significantly enhance the initiation of tumorigenesis. In contrast, the latency of malignancy in p53(+/-) mice was dramatically reduced. We propose that in Mre11(ATLD1/ATLD1) mice, genome instability and cell cycle checkpoint defects reduce viability in early embryos and in proliferating cells, while promoting malignancy in the context of an initiating lesion.     

Structural abnormalities of hippocampus in 101/HY mice

We studied cytoarchitectonics of the hippocampus in 101/HY and CBA mice on brain sections stained after Nissl and Timm. In CBA mice, the structure of hippocampus was normal. In 10/HY mice, stratum pyramidale in field CA3 was "splitted" and the density of pyramidal neurons was decreased. Abnormalities were also found in the zone of suprapyramidal projections of mossy fibers (sp-ME), i.e., terminals of axons of the fascia dentata granular cells on the apical dendrites of pyramids. If in CBA mice the sp-MF zone was normal, i.e., looked like a vast compact formation or dense ordered bundle, in 101/HY mice, the sp-MF zone represented a group of scattered, diffuse, and interrupted bundles of varying length, some of which were incorporated in stratum pyramidale. Possible causes of the described morphological abnormalities are discussed, as well as their relation to specific features of biology, behavior, and neurological status of 101/HY mice.