Epigenetic dysregulation from chromosomal transit in micronuclei

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers [1-4], yet whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei [5, 6], and subsequent micronuclear envelope rupture [7] profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice as well as cancer and non-transformed cells. Some of the changes to histone PTMs occur due to micronuclear envelope rupture whereas others are inherited from mitotic abnormalities prior to micronucleus formation. Using orthogonal techniques, we show that micronuclei exhibit extensive differences in chromatin accessibility with a strong positional bias between promoters and distal or intergenic regions. Finally, we show that inducing CIN engenders widespread epigenetic dysregulation and that chromosomes which transit in micronuclei experience durable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, in addition to genomic copy number alterations, CIN can serve as a vehicle for epigenetic reprogramming and heterogeneity in cancer.

Zika Virus Overview: Transmission, Origin, Pathogenesis, Animal Model and Diagnosis

Zika virus (ZIKV) was first discovered in 1947 in Uganda. ZIKV did not receive substantial attention until Brazil hosted the 2016 Summer Olympic Games, and ZIKV reached a global audience. ZIKV is a flavivirus transmitted chiefly through mosquito bites, sexual intercourse and, to a lesser extent, breastfeeding. The recent discovery of how ZIKV causes congenital neurodevelopmental defects, including microcephaly, has led to reevaluation of the importance of the interaction of ZIKV with centrosome organization, because centrosomes play an important role in cell division. When ZIKV disrupts centrosome organization and mitotic abnormalities, neural progenitor differentiation is altered, thereby resulting in cell cycle arrest, increased apoptosis and inhibition of neural progenitor cell differentiation; subsequently, abnormalities in neural cell development can result in microcephaly. To aid in the understanding of the importance of ZIKV infection, this review article provides an overview of its history, transmission routes, pathogenesis, animal models and diagnosis.

The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development.

The cytological and molecular investigation of the toxic effects of the herbicide Roundup on Cucumis sativus

In the current study, it is aimed to investigate the toxic effects of a widely used herbicide Roundup containing active ingredient glyphosate on cucumber (Cucumis sativus) by cytological and molecular investigation. Three different concentrations (0.6%, 1.2% and 2.4%) of Roundup were applied to cucumber for 48 and 72 hours. At the end of the application procedure, the germination percentage, mean root length, mitotic frequency and mitotic abnormalities, RAPD profiles and Genomic template stability (GTS) were determined in root apical meristematic cells. For RAPD PCR analysis 10 RAPD primers were used, 8 of them produced band patterns and it was found that 5 RAPD primers among them produced unique polymorphic band patterns and subsequently were used to produce a total of 24 bands. Observed percentage of polymorphism was 26%. The changes in RAPD profiles after Roundup treatment was included variations as gain and/or loss of bands compared with the control group. Genomic template stability changed in RAPD profiles at various Roundup concentrations.

Continuous Long-Term Exposure to Low Concentrations of MWCNTs Induces an Epithelial-Mesenchymal Transition in BEAS-2B Cells

In the field of nanotechnology, the use of multi-walled carbon nanotubes (MWCNTs) is growing. Pulmonary exposure during their production, use, and handling is raising concerns about their potential adverse health effects. The purpose of this study is to assess how the physical characteristics of MWCNTs, such as diameter and/or length, can play a role in cellular toxicity. Our experimental design is based on the treatment of human bronchial epithelial cells (BEAS-2B) for six weeks with low concentrations (0.125–1 µg/cm2) of MWCNTs having opposite characteristics: NM-403 and Mitsui-7. Following treatment with both MWCNTs, we observed an increase in mitotic abnormalities and micronucleus-positive cells. The cytotoxic effect was delayed in cells treated with NM-403 compared to Mitsui-7. After 4–6 weeks of treatment, a clear cellular morphological change from epithelial to fibroblast-like phenotype was noted, together with a change in the cell population composition. BEAS-2B cells underwent a conversion from the epithelial to mesenchymal state as we observed a decrease in the epithelial marker E-cadherin and an increased expression of mesenchymal markers N-cadherin, Vimentin, and Fibronectin. After four weeks of recovery, we showed that the induced epithelial-mesenchymal transition is reversible, and that the degree of reversibility depends on the MWCNT.

O-020 Aneuploidy and mosaicism in human embryos: How correct detection may improve IVF clinical outcomes

text Study question Can new next-generation sequencing (NGS) based strategies for preimplantation genetic testing of aneuploidy (PGT-A) improve clinical outcomes after assisted reproductive technology (ART)? Summary answer Recent randomised controlled trials (RCTs) suggest that NGS-based PGT-A strategies can improve clinical outcomes for older women. The clinical management of mosaic embryos remains controversial. What is known already There are two types of chromosome abnormalities present in embryos, meiotic arising mostly during oogenesis, and mitotic arising after fertilisation. Meiotic aneuploidies are present in all of the embryonic cells and in their vast majority are lethal. Conversely, mitotic abnormalities are present in only part of the embryonic cells with the remaining cells having a different cytogenetic constitution. This phenomenon is known as mosaicism. The sensitivity of NGS meant that mosaic aneuploidy became readily detectable in trophectoderm (TE) samples during PGT-A. The viability and clinical management of mosaic embryos has led to debates and controversies in the reproductive medicine field. Study design, size, duration The study involved an assessment of the impact of mosaic chromosome abnormalities to embryonic viability and clinical outcomes after ART cycles using PGT-A via NGS. A large number of embryos generated in IVF clinics in Europe and the USA was examined. Participants/materials, setting, methods Embryos were generated by couples referred for PGT-A due to various indications. All embryos were cultured to the blastocyst stage, and underwent a TE biopsy, followed by vitrification. TE samples were shipped to 6 reference PGT laboratories and analysed via the use of the same NGS platform. Mosaic chromosome abnormalities were scored according to validated thresholds set by the reference laboratories. The clinical management of mosaic embryos took place according to published guidelines. Main results and the role of chance Comparison of clinical outcomes seen after the transfer of mosaic embryos with those seen after the transfer of euploid embryos led to the following findings: Mosaic embryos with <40% abnormal cells in the TE sample had an OIR of 50% compared to 27% for mosaics with 40–80% abnormal cells in the TE, and 9% for complex mosaic embryos. Karyotyping of ongoing pregnancies resulting after the transfer of mosaic embryos demonstrated a normal chromosome constitution of the resulting foetuses. Limitations, reasons for caution - Cytogenetic classification was based on TE samples removed from blastocysts during PGT-A analysis. As only a fraction of the cells from each embryo are tested, inevitably some mosaic embryos will be incorrectly classified fully euploid or aneuploid. However, this misclassification is expected to have little impact on the results. Wider implications of the findings - The transfer of NGS-classified mosaic embryos was associated with poorer clinical outcomes compared to euploid embryos. However, the ongoing pregnancies resulting from mosaic transfers were euploid. NGS’s ability to identify embryos of reduced viability has the potential to improve IVF clinical outcomes.

Human Microcephaly Protein RTTN Is Required for Proper Mitotic Progression and Correct Spindle Position

Autosomal recessive primary microcephaly (MCPH) is a complex neurodevelopmental disorder characterized by a small brain size with mild to moderate intellectual disability. We previously demonstrated that human microcephaly RTTN played an important role in regulating centriole duplication during interphase, but the role of RTTN in mitosis is not fully understood. Here, we show that RTTN is required for normal mitotic progression and correct spindle position. The depletion of RTTN induces the dispersion of the pericentriolar protein γ-tubulin and multiple mitotic abnormalities, including monopolar, abnormal bipolar, and multipolar spindles. Importantly, the loss of RTTN altered NuMA/p150Glued congression to the spindle poles, perturbed NuMA cortical localization, and reduced the number and the length of astral microtubules. Together, our results provide a new insight into how RTTN functions in mitosis.

Telomere erosion in human pluripotent stem cells leads to ATR-mediated mitotic catastrophe

It is well established that short telomeres activate an ATM-driven DNA damage response that leads to senescence in terminally differentiated cells. However, technical limitations have hampered our understanding of how telomere shortening is signaled in human stem cells. Here, we show that telomere attrition induces ssDNA accumulation (G-strand) at telomeres in human pluripotent stem cells (hPSCs), but not in their differentiated progeny. This led to a unique role for ATR in the response of hPSCs to telomere shortening that culminated in an extended S/G2 cell cycle phase and a longer period of mitosis, which was associated with aneuploidy and mitotic catastrophe. Loss of p53 increased resistance to death, at the expense of increased mitotic abnormalities in hPSCs. Taken together, our data reveal an unexpected dominant role of ATR in hPSCs, combined with unique cell cycle abnormalities and, ultimately, consequences distinct from those observed in their isogenic differentiated counterparts.

Deciphering colchicine like actions of clerodin in terms of microtubule destabilization based mitotic abnormalities, G2/M-phase arrest, and plant polyploidy

Clerodin (C24H34O7), a clerodane diterpenoid, is a bitter principle of Clerodendrum viscosum. The present study aimed to decipher colchicine-like actions of clerodin in terms of microtubule destabilization based mitotic abnormalities, G2-M arrest, and plant polyploidy. Purified clerodin showed increased metaphase frequency in Human Peripheral Blood Lymphocytes (HPBLs), Human Embryonic Kidney cells (HEK-293), and Allium cepa root apical meristem cells. Both squashed slide of the onion root tip and flow cytometric analysis of radish protoplast revealed a significantly increased frequency of polyploid cells. Flow cytometric analysis showed an increase in frequencies of G2-M in MCF-7 cells from 6.10 to 16.25% after clerodin (200μg/mL) treatment for 24 h. Confocal microscopy imaging of tubulin in clerodin-treated MCF-7 cells revealed microtubule destabilization. Molecular docking and LIGPLOT analysis indicate that clerodin interact in the colchicine binding site, including, single hydrogen bond with Asn 101 of α-tubulin. In summary, our experimental data revealed that clerodin has metaphase arresting, microtubule destabilization, and polyploidy inducing ability similar to colchicine. Molecular docking analysis revealed for the first time that clerodin and colchicine interact at the common site of tubulin residue indicating a common mechanism of action. The results also indicate similar cytotoxic potentialities of both clerodin and colchicine even though they belong to different chemical groups. Thus, clerodin may be used in place of colchicine as a plant polyploidy inducing agent in plant breeding programs in Agriculture.

DIPG-21. INDUCTION OF MITOTIC ABNORMALITIES AND BMI-1 MODULATION TO TREAT DIFFUSE INTRINSIC PONTINE GLIOMA

Diffuse intrinsic pontine glioma (DIPG) is a poor-prognosis pediatric brain tumor with a median survival of less than one year. No effective therapy is currently available, and no therapeutic advances have been made in several decades. BMI-1 is a member of the multimeric protein complex Polycomb repressor complex 1 (PRC1). It has been implicated in self-renewal of normal and cancer cells, and in DNA damage signaling. We have previously identified BMI-1 as a potential therapeutic target in DIPG and have shown that BMI-1 is highly expressed in DIPG tumors regardless of histone 3 subtype. In the present study, we show that the modulation of BMI-1 leads to DNA damage, M phase cell cycle arrest, chromosome abnormalities and cell death. Furthermore, modulation of BMI-1 sensitizes DIPG patient-derived stem-like cells to ionizing radiation (IR). Treatment of DIPG stem-like cells with PTC596, a BMI-1 modulator, and IR, impairs the kinetics of DNA damage response (DDR). Both DDR foci formation and resolution were delayed, resulting in further reduction in cell viability compared with either treatment alone. In vivo, treatment of mice bearing DIPG xenografts with PTC596 leads to decreased tumor volume and growth kinetics, increased in-tumor apoptosis and sustained animal survival benefit. Gene expression analysis indicates that BMI-1 expression correlates positively with DIPG stemness and BMI-1 signature. Together our findings indicate that BMI-1 modulation is associated with mitotic abnormalities, impaired DDR and cell death, supporting the combination of BMI-1 modulation and radiation as a promising novel therapy to treat children with DIPG.