Mutations in NCAPG2 Cause a Severe Neurodevelopmental Syndrome that Expands the Phenotypic Spectrum of Condensinopathies.

The use of whole-exome and whole-genome sequencing has been a catalyst for a genotype-first approach to diagnostics. Under this paradigm, we have implemented systematic sequencing of neonates and young children with a suspected genetic disorder. Here, we report on two families with recessive mutations in NCAPG2 and overlapping clinical phenotypes that include severe neurodevelopmental defects, failure to thrive, ocular abnormalities, and defects in urogenital and limb morphogenesis. NCAPG2 encodes a member of the condensin II complex, necessary for the condensation of chromosomes prior to cell division. Consistent with a causal role for NCAPG2, we found abnormal chromosome condensation, augmented anaphase chromatin-bridge formation, and micronuclei in daughter cells of proband skin fibroblasts. To test the functional relevance of the discovered variants, we generated an ncapg2 zebrafish model. Morphants displayed clinically relevant phenotypes, such as renal anomalies, microcephaly, and concomitant increases in apoptosis and altered mitotic progression. These could be rescued by wild-type but not mutant human NCAPG2 mRNA and were recapitulated in CRISPR-Cas9 F0 mutants. Finally, we noted that the individual with a complex urogenital defect also harbored a heterozygous NPHP1 deletion, a common contributor to nephronophthisis. To test whether sensitization at the NPHP1 locus might contribute to a more severe renal phenotype, we co-suppressed nphp1 and ncapg2, which resulted in significantly more dysplastic renal tubules in zebrafish larvae. Together, our data suggest that impaired function of NCAPG2 results in a severe condensinopathy, and they highlight the potential utility of examining candidate pathogenic lesions beyond the primary disease locus.

Yeast osmoregulation - glycerol still in pole position.

In response to osmotic dehydration cells sense, signal, alter gene expression, and metabolically counterbalance osmotic differences. The main compatible solute/osmolyte that accumulates in yeast cells is glycerol, which is produced from the glycolytic intermediate dihydroxyacetone phosphate. This review covers recent advancements in understanding mechanisms involved in sensing, signaling, cell-cycle delays, transcriptional responses as well as post-translational modifications on key proteins in osmoregulation. The protein kinase Hog1 is a key-player in many of these events, however, there is also a growing body of evidence for important Hog1-independent mechanisms playing vital roles. Several missing links in our understanding of osmoregulation will be discussed and future avenues for research proposed. The review highlights that this rather simple experimental system-salt/sorbitol and yeast-has developed into an enormously potent model system unravelling important fundamental aspects in biology.

High concentration of extracellular nucleotides suppresses cell growth via delayed cell cycle progression in cancer and noncancer cell lines.

Tumor necrosis frequently occurs in malignant tumors, showing rapid growth and invasion. This phenomenon is generally regarded as simple ischemic necrosis due to insufficient tumor vessels and blood supply. However, the necrotic tissue contains high amount of nuclear substances, DNA, and nucleoproteins that may affect the surrounding tumor cells by promoting or suppressing the tumor cell growth in vivo. This study focused on the effects of an externally administered water-soluble nuclear crude extract (SNE) containing nuclear protein and oligonucleotides on several human cancer and noncancer cell lines. The results demonstrated that the SNE suppressed cell growth in cancer and noncancer cells in vitro. Through the flow cytometry analysis of the nuclear DNA content, it was observed that the SNE increased and decreased cell proportion in the S and G2/M phases, respectively, thereby suggesting that the cell growth inhibition was due to cell cycle delay, and not due to apoptosis. These studies suggest that the high-concentration of extracellular nucleotides generated as a result of tumor necrosis and/or released from infiltrated neutrophils could suppress the growth of surrounding cancer and intrinsic cells, which provides us some insights into an alternative anticancer strategy for patients with highly malignant necrotic tumor.

Endocrine-Independent Cytotoxicity of Bisphenol A Is Mediated by Increased Levels of Reactive Oxygen Species and Affects Cell Cycle Progression.

Bisphenol A (BPA) is used for the production of plastics and epoxy resins, which are part of packaging materials for food and beverages, and can migrate into food and the environment, thus exposing human beings to its effects. Exposure to BPA has been associated with oxidative stress, cell cycle changes, and genotoxicity, and is mediated by its known endocrine-disrupting activity. Possible BPA cytotoxicity without mediation by estrogen receptors has been reported in the literature. Here, we show the toxic effects of BPA by live-cell imaging on the fission yeast Schizosaccharomyces pombe, an experimental model lacking estrogen receptors, which were in line with data from flow cytometry on intracellular oxidation (76.4 ± 14.4 and 19.4 ± 16.1% of fluorescent cells for BPA treatment and control, respectively; p < 0.05) as well as delay in cell cycle progression (after 90 min of experiment, 48.4 ± 4.30 and 64.6 ± 5.46% of cells with a 4C DNA content for BPA treatment and control, respectively; p < 0.05) upon exposure to BPA. These results strongly support the possibilities that BPA-induced cell cycle changes can be independent of estrogen receptors and that live-cell imaging is a powerful tool for genotoxic analysis.

Effect of cell cycle duration on somatic evolutionary dynamics.

Cellular checkpoints prevent damage and mutation accumulation in tissue cells. DNA repair is one mechanism that can be triggered by checkpoints and involves temporary cell cycle arrest and thus delayed reproduction. Repair-deficient cells avoid this delay, which has been argued to lead to a selective advantage in the presence of frequent damage. We investigate this hypothesis with stochastic modeling, using mathematical analysis and agent-based computations. We first model competition between two cell types: a cell population that enters temporary cell cycle arrest, corresponding to repair (referred to as arresting cells), and one that does not enter arrest (referred to as nonarresting cells). Although nonarresting cells are predicted to grow with a faster rate than arresting cells in isolation, this does not translate into a selective advantage in the model. Interestingly, the evolutionary properties of the nonarresting cells depend on the measure (or observable) of interest. When examining the average populations sizes in competition simulations, nonarresting and arresting cells display neutral dynamics. The fixation probability of nonarresting mutants, however, is lower than predicted for a neutral scenario, suggesting a selective disadvantage in this setting. For nonarresting cells to gain a selective advantage, additional mechanisms must be invoked in the model, such as small, repeated phases of tissue damage, each resulting in a brief period of regenerative growth. The same properties are observed in a more complex model where it is explicitly assumed that repair and temporary cell cycle arrest are dependent on the cell having sustained DNA damage, the rate of which can be varied. We conclude that repair-deficient cells are not automatically advantageous in the presence of frequent DNA damage and that mechanisms beyond avoidance of cell cycle delay must be invoked to explain their emergence.

Identification of a new gene regulatory circuit involving B cell receptor activated signaling using a combined analysis of experimental, clinical and global gene expression data.

To discover new regulatory pathways in B lymphoma cells, we performed a combined analysis of experimental, clinical and global gene expression data. We identified a specific cluster of genes that was coherently expressed in primary lymphoma samples and suppressed by activation of the B cell receptor (BCR) through αIgM treatment of lymphoma cells in vitro. This gene cluster, which we called BCR.1, includes numerous cell cycle regulators. A reduced expression of BCR.1 genes after BCR activation was observed in different cell lines and also in CD10+ germinal center B cells. We found that BCR activation led to a delayed entry to and progression of mitosis and defects in metaphase. Cytogenetic changes were detected upon long-term αIgM treatment. Furthermore, an inverse correlation of BCR.1 genes with c-Myc co-regulated genes in distinct groups of lymphoma patients was observed. Finally, we showed that the BCR.1 index discriminates activated B cell-like and germinal centre B cell-like diffuse large B cell lymphoma supporting the functional relevance of this new regulatory circuit and the power of guided clustering for biomarker discovery.

Application of cell sorting for enhancing the performance of the cytokinesis-block micronucleus assay.

Among the numerous methods available to assess genotoxicity, the cytokinesis-block micronucleus (CBMN) assay is very popular due its relative simplicity and power to detect both clastogenic and aneugenic compounds. A problem with the CBMN assay is that all DNA damaging agents also inhibit the ability of cells to progress through mitosis, leading to a low number of binucleated cells (BNCs). One method to resolve this issue is to ensure a sufficient proportion of BNCs in the samples. In the current study, the applicability of a cell sorting system capable of isolating cell fractions containing abundant BNCs was investigated. Furthermore, to investigate the relationship between the cell division delay due to radiation exposure and the generation of BNCs and micronuclei (MN), we assessed a series of lag times between radiation exposure and addition of cytochalasin-B (Cyt-B). Cells from the human chronic myelogenous leukemia cell line K562 were exposed to X-rays (2 Gy and 4 Gy), and Cyt-B was subsequently added at 0, 6 and 12 h following irradiation. After treatment with Cyt-B for 24 h, the percentage of BNCs, the MN frequency and the cell cycle distribution were analyzed. In addition, cells displaying the DNA contents corresponding to BNCs were isolated and analyzed. The results indicate that applying the cell sorter to the CBMN assay increased the percentage of BNCs compared with the standard method. Thus, this technique is a promising way of enhancing the capacity of the CBMN assay.

Induction of sister chromatid exchanges and cell division delays by clomiphene citrate in human lymphocytes.

OBJECTIVE: Clomiphene citrate (CC) is a selective estrogen receptor modulator and is used for the treatment of in vitro fertilization, intracytoplasmic sperm injection, intrauterine insemination, and so on. In this study, sister chromatid exchanges (SCEs) and cell cycle delays were analyzed to investigate genotoxicity and cytotoxicity of CC in peripheral blood lymphocytes of healthy women. METHODS: Human peripheral blood lymphocytes obtained from two donors were used to detect genotoxicity and cytotoxicity of CC. Lymphocytes were treated with various concentrations (0.40, 0.80, 1.60, and 3.20 µg/ml) of CC. A negative (distilled water) and a positive control (mitomycin-C = 0.20 µg/ml) were also used simultaneously with test substance-treated cultures. SCEs and cell division delays were measured from 25 cells and 100 cells perdonor, respectively. RESULTS: CC significantly increased the mean SCE value at all concentrations compared with the negative control. This increase was found to be dose dependent (r = 0.83) and at the highest concentration, nearly two times higher increase was observed than the negative control. However, replication index was not affected by the CC treatment. CONCLUSION: The present study shows that CC is genotoxic for human lymphocytes in vitro. Further investigations, especially in vivo are now needed in different test organisms to clarify the genotoxic activity of CC, which should also help to better understand genotoxic mechanism of this ovulation-stimulating drug.

Impact of cell cycle delay on micronucleus frequency in TK6 cells.

Previous studies with TK6 cells have shown that extending the recovery period after pulse treatment allows for greater micronucleus expression for some compounds. This study explores the role of cell cycle delay in micronucleus expression after pulse treatment with three model genotoxins [mitomycin C, etoposide (ETOP), vinblastine]. Cells were treated for 4 hr and allowed to recover for 36 hr with samples removed at various time points during the recovery period and analyzed for cell cycle distribution, apoptosis and micronucleus frequency. Our results show that mitomycin C causes cell cycle delay for 20 hr after pulse treatment and cell cycle perturbation is no longer evident after 36 hr of recovery. The micronucleus frequency of cells sampled at 36 hr is doubled when compared with cells sampled at 20 hr after mitomycin C removal. When cells were treated with indirect acting genotoxins (ETOP, vinblastine), cell cycle perturbation was not observed at the 20 hr time point. Micronucleus frequency after treatment with either ETOP or vinblastine did not differ between the 20 hr and the 36 hr time point. All three compounds induced similar levels of apoptosis ranging from 4.5 to 5.6% with maximum induction occurring at the 36-hr time point. We conclude that TK6 cells exhibit extended cell cycle arrest after exposure to MMC and can go on to express micronuclei, after overcoming cell cycle arrest.