Nuclear p120 catenin is a component of the perichromosomal layer and coordinates sister chromatid segregation during mitosis in lung cancer cells.

Abnormal expression of p120 catenin is associated with the malignant phenotype in human lung cancer. Numerous studies have focused on the function of p120 catenin in the juxta-membrane compartment. However, the role of nuclear p120 catenin remains unclear. In this study, the dynamic changes in nuclear p120 catenin localization during cell cycle progression were investigated. Immunofluorescent staining, FACS analysis, and western blotting revealed that nuclear p120 catenin is a major architectural constituent of the chromosome periphery during mitosis. During mitosis, granule-like p120 catenin dispersed into a cloudy-like structure and formed cordon-like structures surrounding the condensed chromosomes to create the peri-chromosomal layer. Interestingly, lumican and p120 catenin colocalized at the spindle fiber where the perichromosomal layer connects to the condensed chromosomes during mitosis. Furthermore, downregulation of p120 catenin using a specific siRNA induced cell cycle stalling in the G2/M phase and promoted aneuploidy. This study validates the role of nuclear p120 catenin in the formation of the chromosome periphery and reveals the p120 catenin-lumican interaction may couple orientation of cell division with the segregation of sister chromatids during mitosis. Our data suggest the protective role of p120 catenin in maintaining the integrity of chromosomes, and also warrants further studies to evaluate the contribution of the loss of p120 catenin to the creation of gene rearrangement in cancer evolution and tumor progression.

Identification of Different miRNAs and Their Relevant miRNA Targeted Genes Involved in Sister Chromatid Cohesion and Segregation (SCCS)/chromatin Remodeling Pathway on T1G3 Urothelial Carcinoma (UC) Response to BCG Immunotherapy.

BACKGROUND: Till now, no definite clinical or laboratory marker can predict the recurrence or progression of T1 G3 urothelial carcinoma (UC). Genetic aberrations of the chromatin remodeling genes and sister chromatid cohesion and segregation (SCCS) were identified in UC. Here we investigated the impact of novel miRNAs and their targeted expressed SCCS and chromatin remodeling genes on T1G3 UC response to Bacillus Calmette-Guérin (BCG) therapy. METHODS: One hundred tissue samples were obtained from NMIBC patients. Gene expression and immunohistochemical assay of STAG2, ARID1A, NCOR1and UTX were assessed. MiRNA analysis for their targeting miRNAs (miR-21, miR-31, Let7a and miR-199a) was carried out. Assessed genes were compared between responders and no responders to BCG. Univariate and multivariate analysis of predictors of disease recurrence and progression were performed using cox regression analysis. RESULTS: Thirty-two and 22 patients developed recurrence and progression to MIBC (BCG non-responders). BCG non-responders showed statistically significant higher expression of miR-21 and their targeted STAG2, miR-199a and NCOR1 gene (P < .001), and lower expression of miR-31, Let7a, ARID1A and UTX genes (P < .001). Higher miR-199a (P = .006) and lower miR-31 (P = .01), ARID1A (P = .008) and UTX (P = .03) were independent predictor of higher tumor recurrence. Recurrent disease (P = .003), higher expression of STAG2 (P = .01), NCOR1 (P = .01) and miR-21 (P = .03) genes and lower expression of miR-31 (P = .02), Let7a (P = .04) and ARID1A (P = .04) genes were the independent predictor of disease progression. CONCLUSION: Upregulation of STAG2 and NCOR1 and down regulation of ARID1A and UTX genes and their targeting miRNAs were associated with UC non-response to BCG.

Role of the DDX11 DNA Helicase in Warsaw Breakage Syndrome Etiology.

Warsaw breakage syndrome (WABS) is a genetic disorder characterized by sister chromatid cohesion defects, growth retardation, microcephaly, hearing loss and other variable clinical manifestations. WABS is due to biallelic mutations of the gene coding for the super-family 2 DNA helicase DDX11/ChlR1, orthologous to the yeast chromosome loss protein 1 (Chl1). WABS is classified in the group of "cohesinopathies", rare hereditary diseases that are caused by mutations in genes coding for subunits of the cohesin complex or protein factors having regulatory roles in the sister chromatid cohesion process. In fact, among the cohesion regulators, an important player is DDX11, which is believed to be important for the functional coupling of DNA synthesis and cohesion establishment at the replication forks. Here, we will review what is known about the molecular and cellular functions of human DDX11 and its role in WABS etiopathogenesis, even in light of recent findings on the role of cohesin and its regulator network in promoting chromatin loop formation and regulating chromatin spatial organization.

Chromosome instability induced by a single defined sister chromatid fusion.

Chromosome fusion is a frequent intermediate in oncogenic chromosome rearrangements and has been proposed to cause multiple tumor-driving abnormalities. In conventional experimental systems, however, these abnormalities were often induced by randomly induced chromosome fusions involving multiple different chromosomes. It was therefore not well understood whether a single defined type of chromosome fusion, which is reminiscent of a sporadic fusion in tumor cells, has the potential to cause chromosome instabilities. Here, we developed a human cell-based sister chromatid fusion visualization system (FuVis), in which a single defined sister chromatid fusion is induced by CRISPR/Cas9 concomitantly with mCitrine expression. The fused chromosome subsequently developed extra-acentric chromosomes, including chromosome scattering, indicative of chromothripsis. Live-cell imaging and statistical modeling indicated that sister chromatid fusion generated micronuclei (MN) in the first few cell cycles and that cells with MN tend to display cell cycle abnormalities. The powerful FuVis system thus demonstrates that even a single sporadic sister chromatid fusion can induce chromosome instability and destabilize the cell cycle through MN formation.

Long-term remission of bilateral Wilms tumors that developed from premature separation of chromatids/mosaic variegated aneuploidy syndrome due to bilateral nephrectomy and peritoneal dialysis.

We report a 38-month-old Japanese male with premature chromatid separation/mosaic variegated aneuploidy syndrome bearing biallelic BUB1B germline mutations who suffered from bilateral Wilms tumor. After right nephrectomy, dactinomycin monotherapy was administered for the left Wilms tumor; however, severe adverse reaction prevented the patient from receiving further chemotherapy. Left nephrectomy was then performed without postoperative chemotherapy. The patient survived for 15 months after bilateral nephrectomy without peritoneal relapse, metastasis of Wilms tumor, or the occurrence of rhabdomyosarcoma and maintained a good quality of life while receiving peritoneal dialysis at home.

Differential in vivo genotoxicity of arsenic trioxide in glutathione depleted mouse bone marrow cells: expressions of Nrf2/Keap1/P62.

Generation of reactive oxygen species is one of the major contributors in arsenic-induced genotoxicity where reduced glutathione (GSH) could be an important determining factor. To understand the role of endogenous GSH, arsenic trioxide (As2O3) was administered in buthionine sulfoximine (BSO)- and N-acetyl-L-cysteine (NAC)-treated mice. As2O3-induced significant chromosome aberrations (CAs) in all treatment groups compared with the control. BSO-treated mouse bone marrow cells showed significant CAs at a dose of 2 mg As2O3 kg(-1) b.w. Similar induction was not evident at 4 mg As2O3 kg(-1) b.w. and exhibited antagonistic effect at 8 mg As2O3 kg(-1) b.w. To understand this differential effect, expression pattern of Nrf2 was observed. Nrf2 expression increased following As2O3 treatment in a dose-dependent manner up to 4 mg As2O3 kg(-1) b.w after which no further increase was noticed. NAC pre-treatment significantly reduced the extent of As2O3-induced CAs suggesting the protective role of endogenous GSH against arsenic-induced genotoxicity.

Sister chromatid cohesion defects are associated with chromosome instability in Hodgkin lymphoma cells.

BACKGROUND: Chromosome instability manifests as an abnormal chromosome complement and is a pathogenic event in cancer. Although a correlation between abnormal chromosome numbers and cancer exist, the underlying mechanisms that cause chromosome instability are poorly understood. Recent data suggests that aberrant sister chromatid cohesion causes chromosome instability and thus contributes to the development of cancer. Cohesion normally functions by tethering nascently synthesized chromatids together to prevent premature segregation and thus chromosome instability. Although the prevalence of aberrant cohesion has been reported for some solid tumors, its prevalence within liquid tumors is unknown. Consequently, the current study was undertaken to evaluate aberrant cohesion within Hodgkin lymphoma, a lymphoid malignancy that frequently exhibits chromosome instability. METHODS: Using established cytogenetic techniques, the prevalence of chromosome instability and aberrant cohesion was examined within mitotic spreads generated from five commonly employed Hodgkin lymphoma cell lines (L-1236, KM-H2, L-428, L-540 and HDLM-2) and a lymphocyte control. Indirect immunofluorescence and Western blot analyses were performed to evaluate the localization and expression of six critical proteins involved in the regulation of sister chromatid cohesion. RESULTS: We first confirmed that all five Hodgkin lymphoma cell lines exhibited chromosome instability relative to the lymphocyte control. We then determined that each Hodgkin lymphoma cell line exhibited cohesion defects that were subsequently classified into mild, moderate or severe categories. Surprisingly, ~50% of the mitotic spreads generated from L-540 and HDLM-2 harbored cohesion defects. To gain mechanistic insight into the underlying cause of the aberrant cohesion we examined the localization and expression of six critical proteins involved in cohesion. Although all proteins produced the expected nuclear localization pattern, striking differences in RAD21 expression was observed: RAD21 expression was lowest in L-540 and highest within HDLM-2. CONCLUSION: We conclude that aberrant cohesion is a common feature of all five Hodgkin lymphoma cell lines evaluated. We further conclude that aberrant RAD21 expression is a strong candidate to underlie aberrant cohesion, chromosome instability and contribute to the development of the disease. Our findings support a growing body of evidence suggesting that cohesion defects and aberrant RAD21 expression are pathogenic events that contribute to tumor development.

Age-related changes in genomic stability of horses.

Recently, the old horse has been proposed as a model to study telomere-dependent senescence, immunosenescence and inflamm-aging. In the present paper, we used 80 Hucul and Anglo-Arabian horses divided into 3 age groups (juvenile, adult, old) to evaluate age-dependent changes at the genomic and DNA level and in cell proliferative potential. The level of positive TUNEL cells (both apoptotic and with DNA fragmentation), oxidative DNA damage (8-oxoG immunostaining), sister chromatid exchange and bleomycin-induced chromatid breaks were significantly increased in the combined old group compared to the combined adult group. We observed a negative correlation between micronuclei formation and age, which may be associated with damaged cells undergoing apoptosis, rather than expressing micronuclei. We were unable to show any significant changes in the nuclear division index value, which reflects the proliferative status of the viable cell fraction during aging. Here, we show that breed-independent and age-associated changes in genomic stability may contribute, at least in part, to the aging process in the horse.

The cytogenetics of polar bodies: insights into female meiosis and the diagnosis of aneuploidy.

Female meiosis is comprised by two cell divisions, meiosis I (MI) and II (MII) and two different stages at which the development of the oocyte is temporarily halted. In the case of MI, this pause can potentially last for four to five decades. This added layer of complexity distinguishes female gametogenesis from its male counterpart. The single most important genetic factor impacting human reproductive success is aneuploidy. Aneuploid embryos may undergo permanent arrest during preimplantation development, fail to implant or spontaneously abort. Most aneuploidies originate during female meiosis and become increasingly common with advancing maternal age. To shed further light on the nature of aneuploidy in human oocytes, we utilized comparative genomic hybridization (CGH) to provide a detailed cytogenetic analysis of 308 first and second polar bodies (PBs). These were biopsied from fertilized oocytes, generated by 70 reproductively older women (average maternal age of 40.8 years). The total oocyte abnormality rate was 70%, and MII anomalies predominated over MI (50% aneuploidy rate versus 40.3%). Both whole chromosome non-disjunction and unbalanced chromatid predivision were seen, but the latter was the dominant MI aneuploidy-causing mechanism. Chromosome losses occurred more frequently than chromosome gains, especially during MI. Chromosomes of all sizes were found to participate in aneuploidy events, although errors involving smaller chromosomes were more common. These data reveal the spectrum of aneuploidies arising after each meiotic division, indicating that oocyte-derived abnormalities present at conception differ from those observed in established pregnancies. It is also clear that advancing maternal age had a significant adverse effect on female meiosis, and that this effect is most pronounced in MII. Indeed, our data suggest that MII may be more susceptible to age-related errors than MI.

Molecular mechanisms and function of the spindle checkpoint, a guardian of the chromosome stability.

For equal segregation, chromosomes, which are distributed randomly in the nucleus of interphase, must be aligned at the spindle equator in mitosis before the onset of sister chromatid separation. The spindle checkpoint is a surveillance mechanism that delays the onset of sister chromatid separation while each chromosome is on the way to the spindle equator. Failure in the function of the checkpoint results in aneuploidy/polyploidy, which would be a cause of cancer. Here, we review chromosome dynamics in mitosis, molecular mechanisms of the spindle checkpoint and finally tumorigenesis triggered by missegregation of chromosomes.

Premature chromatid separation is not a useful diagnostic marker for Cornelia de Lange syndrome.

Cornelia de Lange syndrome (CdLS) is a rare, multiple congenital anomaly/mental retardation syndrome characterized by clinical variability and caused by mutations in the NIPBL (50-60%), SMC1L1 and SMC3 genes (5%), which encode for proteins involved in sister chromatid cohesion. Almost all of the studies of premature chromatid separation (PCS) in CdLS patients have failed to demonstrate that it is specific to CdLS, thus making its diagnostic use controversial. In order to verify the diagnostic usefulness of PCS screening in CdLS, we analysed metaphase spreads from 29 CdLS patients and 24 controls using a rigorous protocol to induce PCS, and precise criteria to score the affected chromosomes. Following exclusion of significant intra-sample variation we scored under blind conditions 150 spreads from a single preparation of each case and computed the ratio between the number of prematurely separated chromatids and the total number of chromatids. The results indicate the extreme variability of PCS in both cohorts (CdLS: mean 2.8 +/- 2.8%; controls: mean 4.0 +/- 5.4%) and highlight the difficulty of PCS monitoring, especially when selecting the control population. The absence of any difference in the frequency of PCS between the patients and controls, or between patients with different clinical or genetic backgrounds, precludes its potential use as an additional diagnostic tool.

Monoallelic BUB1B mutations and defective mitotic-spindle checkpoint in seven families with premature chromatid separation (PCS) syndrome.

Cancer-prone syndrome of premature chromatid separation (PCS syndrome) with mosaic variegated aneuploidy (MVA) is a rare autosomal recessive disorder characterized by growth retardation, microcephaly, childhood cancer, premature chromatid separation of all chromosomes, and mosaicism for various trisomies and monosomies. Biallelic BUB1B mutations were recently reported in five of eight families with MVA syndrome (probably identical to the PCS syndrome). We here describe molecular analysis of BUB1B (encoding BubR1) in seven Japanese families with the PCS syndrome. Monoallelic BUB1B mutations were found in all seven families studied: a single-base deletion (1833delT) in four families; and a splice site mutation, a nonsense mutation, and a missense mutation in one family each. Transcripts derived from the patients with the 1833delT mutation and the splice site mutation were significantly reduced, probably due to nonsense-mediated mRNA decay. No mutation was found in the second alleles in the seven families studied, but RT-PCR of BUB1B and Western blot analysis of BubR1 indicated a modest decrease of their transcripts. BubR1 in the cells from two patients showed both reduced protein expression and diminished kinetochore localization. Their expression level of p55cdc, a specific activator of anaphase-promoting complex, was normal but its kinetochore association was abolished. Microcell-mediated transfer of chromosome 15 (containing BUB1B) into the cells restored normal BubR1 levels, kinetochore localization of p55cdc, and the normal responses to colcemid treatment. These findings indicate the involvement of BubR1 in p55cdc-mediated mitotic checkpoint signaling, and suggest that >50% decrease in expression (or activity) of BubR1 is involved in the PCS syndrome.

Chromosomal aberrations in peripheral lymphocytes of patients treated with radium-224 for ankylosing spondylitis.

The aim of this study was to investigate the in vivo frequency of chromosomal aberrations (primarily dicentric chromosomes and chromatid breaks) potentially induced by (224)Ra alpha-radiation in peripheral lymphocytes. The study was designed to serve as a cytogenetic analysis along with the therapeutic procedure of ankylosing spondylitis patients who were undergoing a treatment with (224)Ra-chloride. The total administered activity was 10 MBq, and the treatment followed a schedule of 10 i.v. injections per week, each with a dose of 1 MBq of (224)Ra. The calculation of absorbed doses delivered to the blood used the models suggested by the ICRP and yielded a value of 4.7 mGy/MBq. The frequency of chromosomal aberrations observed during the course of therapy was related to the blood dose. The frequency of dicentric chromosomes induced in vivo was found to agree well with the corresponding value of dicentrics induced in vitro. However--given that peripheral lymphocytes are in the cell cycle's G(0) stage--an unexpected increase with dose in the yield of chromatid breaks was observed, with about 95% of them occurring in cells without any other chromosome-type aberrations. Reasons for the production of chromatid breaks are discussed.

Characteristics of chromosome instability in the human lymphoblast cell line WTK1.

The characteristics of spontaneous and radiation-induced chromosome instability were determined in each of 50 individual clones isolated from control populations of human lymphoblasts (WTK1), as well as from populations of these cells previously exposed to two different types of ionizing radiation, Fe-56 and Cs-137. The types of chromosome instability did not appear to change in clones surviving radiation exposure. Aneuploidy, polyploidy, chromosome dicentrics and translocations, and chromatid breaks and gaps were found in both control and irradiated clones. The primary effect of radiation exposure was to increase the number of cells within any one clone that had chromosome alterations. Chromosome instability was associated with telomere shortening and elevated levels of apoptosis. The results suggest that the proximal cause of chromosome instability is telomere shortening.

Chromosomal instability syndrome of total premature chromatid separation with mosaic variegated aneuploidy is defective in mitotic-spindle checkpoint.

Skin fibroblast cells from two unrelated male infants with a chromosome-instability disorder were analyzed for their response to colcemid-induced mitotic-spindle checkpoint. The infants both had severe growth and developmental retardation, microcephaly, and Dandy-Walker anomaly; developed Wilms tumor; and one died at age 5 mo, the other at age 3 years. Their metaphases had total premature chromatid separation (total PCS) and mosaic variegated aneuploidy. Mitotic-index analysis of their cells showed the absence of mitotic block after the treatment with colcemid, a mitotic-spindle inhibitor. Bromodeoxyuridine-incorporation measurement and microscopic analysis indicated that cells treated with colcemid entered G1 and S phases without sister-chromatid segregation and cytokinesis. Preparations of short-term colcemid-treated cells contained those cells with chromosomes in total PCS and all or clusters of them encapsulated by nuclear membranes. Cell-cycle studies demonstrated the accumulation of cells with a DNA content of 8C. These findings indicate that the infants' cells were insensitive to the colcemid-induced mitotic-spindle checkpoint.

Mosaic variegated aneuploidy with multiple congenital abnormalities: homozygosity for total premature chromatid separation trait.

Separation of chromatids of all mitotic chromosomes, here called total premature chromatid separation (total PCS), was observed in 67 to 87.5% of repeated cultures of peripheral blood lymphocytes from two unrelated infants. Also noted was a variety of mosaic aneuploidies, especially trisomies, double trisomies, and monosomies, to be called mosaic variegated aneuploidy. The infants both showed severe pre- and postnatal growth retardation, profound developmental retardation, uncontrollable seizures, severe microcephaly, hypoplasia of the brain, Dandy-Walker anomaly, abnormal facial appearance, and bilateral cataract. Patient 1, a girl, in addition had a cleft palate, multiple renal cysts, and Wilms tumor of the left kidney. Whereas patient 2, a boy, had ambiguous external genitalia. They both died within 2 years of age. In the two families of the infants, their parents and three other members showed 2.5 to 47% lymphocytes with total PCS but without mosaic variegated aneuploidy or phenotypic abnormalities. Another 10 relatives studied showed 0 to 1% cells with total PCS and so were judged negative for the total PCS trait. It was deduced that the total PCS trait in the two families was transmitted in an autosomal-dominant fashion, and the two affected infants were homozygous for the trait.

Preimplantation diagnosis of common aneuploidies by the first- and second-polar body FISH analysis.

PURPOSE: A low pregnancy rate in in vitro fertilization (IVF) patients of advanced maternal age may be caused by aneuploidies originating from non disjunction in the first or second meiotic divisions. We introduced genetic testing of oocytes by sampling and fluorescent in situ hybridization (FISH) analysis of the first and second polar bodies, to avoid fertilization and transfer of aneuploid oocytes in IVF patients of advanced maternal age. METHODS: Three hundred and sixty-three IVF patients 34 years and older participated in the study. Using micromanipulation procedures, the first and second polar bodies were removed following their extrusion from the oocytes and studied by FISH, using probes specific for chromosomes 13, 18, and 21 to detect oocytes with common aneuploidies. RESULTS: Of a total of 538 IVF cycles, 3250 oocytes were available for FISH analysis, with conclusive FISH results in 2742 oocytes (84.3%). As many as 1102 (40%) of oocytes were predicted to be aneuploid and not transferred. Of 1640 embryos predicted to be normal, 1145 were transferred in 467 treatment cycles, resulting in 107 pregnancies (23%), from which 67 healthy children have been born, 32 pregnancies spontaneously aborted, and 15 pregnancies are ongoing after being confirmed normal by prenatal diagnosis. CONCLUSIONS: Preimplantation diagnosis by first- and second-polar body FISH analysis allows us to avoid the age-related risk of common aneuploidies in IVF patients of advanced maternal age.

Chromosomal aberrations induced by defined DNA double-strand breaks: the origin of achromatic lesions.

The mechanisms of formation of chromosomal aberrations are poorly understood, despite the common use of aberrations as a measure of the genetic effects of physical and chemical agents. We have used restriction endonucleases to introduce defined DNA double-strand breaks into mammalian cells, and measured chromosomal aberration formation relative to the activity of the endonuclease. The endonucleases AluI and Sau3AI remain active for a relatively short time under simulated cellular conditions and induce achromatic lesions ('gaps') in chromatids only within the first hour or two following treatment. In contrast, the endonuclease MboI (an isoschizomer of Sau3AI) is active for an extremely long time and continues to produce chromatid gaps during the whole 12 hr sampling period. This observation strongly suggests that the aberrations classified as gaps are a manifestation of unrejoined DNA double-strand breaks. The formation of gaps may relate to the opportunities for repair of DNA breaks in relation to cell-cycle position. It is more difficult to relate the formation of structural chromatid aberrations to the endonuclease activity, although at relatively low concentrations all 3 endonucleases gave similar levels of structural aberrations.

Induction of chromosomal aberrations in bone marrow cells of asbestotic rats.

In the present study, cytogenetic effects of Indian chrysotile asbestos in rat bone marrow cells after 290 days of intratracheal inoculation (5 mg dust/0.5 ml normal saline), when it develops massive pulmonary fibrosis, were investigated. The pulmonary fibrosis was confirmed by both histopathological studies and increased collagen content in the lung of the treated animals. In the asbestotic rats a significant increase in chromosomal aberrations was recorded and a decrease in mitotic index of bone marrow cells. The types of chromosomal aberrations in these cells were chromatid gaps and breaks. The results indicate the significant cytogenetic changes in the bone marrow cells of asbestotic rats and also suggest that these changes directly or indirectly may be one of the biological events involved in eliciting the asbestos-mediated toxic responses.