Diploid Cell Line Research Articles

Novel cell line derived from natural host Apodemus Peninsulae for in-vitro studies of tick-borne encephalitis virus

Purpose: The purpose of the research was to develop a natural host-derived cell line for the study of the host specificity of tick-borne encephaitis virus and evalute the reproduction of virus in this cell line. Methods & Materials: The cell line was established from trypsinized homogenates of kidneys of female Apodemus peninsulae (Korean field mouse). Cell line was characterized by cariotyping and by nucleotide sequence analysis of the cytochrome B gene and the D-loop region of mitochondrial genome. Cells were infected with the tick-borne encephalitis virus (TBEV) isolate 92 M belonging to Siberian subtype. Tissue cultures were sampled at 0, 4, 8, 12, 16, 20 and 24 hours (hpi) as well as after 4 and 7 days post infection (dpi). TBEV reproduction dymamics was evaluated by plaque titration of supernatant medium from infected cells. Replication of intracellular RNA was measured by quantitative PCR. Production of TBEV antigen was evaluated using ELISA test. Porcine embryo kidney cell line SPEV was used in parallel as reference. All experiments were performed in triplicates. Results: The adhesive diploid cell line was obtained by serial passages of primary culture and designated as ApnK. Sequencing confirmed the species origin of the cells. This cell line was used for experiments at passage 30. The growth of TBEV infectivity started at 12 hpi and concentration of infectious virus gradually increased up to 4 dpi. Afterwards it stabilized with titres of 7 lg PFU/ml. The synthesis of intracellular genomic RNA was firstly confirmed at 8 hpi and the dynamics was similar to the infectivity growth reaching the plateau at 4 dpi with concentration of 8 lg copies/mkl. TBEV antigen was detected in cell supernatants at 20 hpi and the dynamics corresponded to those of infectivity growth and RNA replication. In all experiments the reproduction of TBEV in ApnK cells was slowly than in SPEV cells up to 4 dpi when they equilibrated. Conclusion: The stabile cell line of natural host of TBEV, A. peninsulae, was established. This cell line susceptible for TBEV infection, however the reproduction of the virus is restricted in comparison to convenient in vitro model, porcine embryo kidney cell line SPEV

Исследование антивирусной активности Глутоксима при инфицировании клеточной линии фибробластов вирусом везикулярного стоматита

New strategy for the treatment of animal infectious diseases is based upon the modulation of the host immune response in order to enhance the clearance of infectious agents and reduce the damaging effects of inflammation in the tissues. The modern approach to the use of immunomodulators (IMD) in veterinary practice consists in the usage of such drugs, which are not only immunomodulating, but also have antiviral, antioxidant, anti-inflammatory, hemostimulating and/or other important properties. The aim of the study was to identify possible antiviral activity of known IMD Glutoxim (GLT) during infection of diploid fibroblast cell lines M-8 and M-22 with vesicular stomatitis virus (VSV). Materials and methods: VSV, strain Indiana, was used. Antiviral activity of GLT investigated: 1) at doses recommended for experiments in vitro: 1, 4 and 8 µg/ml; 2) at low doses: 0,1; 0,25 and 0,5 µg/ml. GLT was added to the cell monolayer according to preventive (for 24 hours prior to VSV infection of cells) and treatment (unanimous with VSV infection) protocols. The antiviral activity of GLT was assessed by the following criteria: ability of the drug to prevent the development of virus cytopathic action, to inhibit the reproduction of VSV, and by expessing virucidal action. Results: GLT in doses recommended for in vitro experiments (1, 4, 8 µg/ml) did not delay the development of a specific virus-induced cytopathic action. The VSV titers in infected cells in the presence of GLT did not differ from those in the control cell lines infected with VSV without the addition of GLT. The latter had no virucidal effect against the VSV. Inoculation of GLT into the cell culture at low doses of 0.1, 0.25 and 0.5 mg/ml led to a significant (more than 100-fold) inhibition of VSV replication 24 hours after infection of cells. At later stages, 40 and 48 hours following infection, the antiviral effect of GLT was not detected. Thus, we established that GLT possesses antiviral effect in vitro, which is manifested 24 hours following infection of diploid fibroblast cell lines with VSV.

Use DNA fingerprinting to identify and confirm triploid embryos in whole genome next generation sequencing (NGS) preimplantation genetic screen (PGS)

Whole genome next generation sequencing (NGS) for preimplantation genetic screen (PGS) has higher sensitivity and accuracy in detecting unbalanced translocation and partial aneuploid than targeted NGS. Whole genome NGS also detect some triploid, the most common polyploidy, if the sex chromosomes are unbalanced (69, XXY, and 69, XYY). Unlike targeted NGS, whole genome NGS unusually does not detect triploid 69, XXX, neither does it provide additional data to confirm triploid 69, XXY and 69, XYY identified in PGS. Here we tested using DNA fingerprinting in combination with whole genome NGS to identify and confirm sex chromosome balanced and unbalanced triploid in PGS. Feasibility and validation study. The feasibility is tested on of 5 known triploid cell lines and 5 known diploid cell lines. 24 fingerprinting loci were tested. Further validation and accuracy were tested on 16 blastocyst biopsy samples whole genome amplification product. All 16 samples had whole genome NGS for PGS and were potential triploid samples. 15 of them were identified by unbalanced sex chromosomes for PGS. 1 of them showed 46, XX for PGS but is suspected to be 69, XXX triploid because of the enlarged nuclei observed by the embryologist. DNA fingerprinting was performed on the 16 PGS samples to identify and confirm the triploid embryos. FISH was performed to confirm the DNA fingerprinting result. Homozygous or heterozygous loci (24 of 24) in DNA fingerprinting were detected for all 5 known diploid cells lines, confirming diploid. For the 5 known triploid cell lines, 5 to 10 out of the 24 DNA fingerprinting loci has 3 genotypes, confirming triploid. Among the 15 potential triploid samples identified by PGS, 13 showed triploid in DNA fingerprinting, with 3 to 11 loci having 3 genotypes; 2 showed diploid in DNA fingerprinting. The enlarged nuclei sample with 46, XX for PGS also showed triploid in DNA fingerprinting, with 7 out of 24 loci having 3 genotypes. The 16 embryos FISH result confirmed the DNA fingerprinting result, demonstrating DNA fingerprinting is accurate in identifying and confirming triploid in PGS sample. DNA fingerprinting test on whole genome amplified PGS samples is a simple and accurate method to examine triploid status of embryos. It can confirm triploid indicated by embryo morphology. Triploid identification in PGS based on unbalance sex chromosomes can be difficult and not reliable, especially when embryo DNA quality is compromised. In this situation DNA fingerprinting can identify and confirm the real triploid embryos. Used in combination with whole genome NGS, DNA fingerprinting allows patients to benefit from the advantages of the whole genome NGS, and at the same time, identifying the triploid embryos to reduce pregnancy loss.

Ploidy-dependent change in cyclin D2 expression and sensitization to cdk4/6 inhibition in human somatic haploid cells

Near-haploidy is observed in certain cancer types, but ploidy-dependent alterations in gene regulation in the haploid state remain elusive. Here, by comparative transcriptome analysis between human isogenic haploid and diploid cell lines, we found lowering of cyclin D2 level in haploids. Acute genome duplication in haploids restored cyclin D2 expression to diploid level, indicating that the regulation of cyclin D2 expression is directly linked to ploidy. Downstream pathways of cyclin D2, such as Rb phosphorylation and p27 sequestration remained intact in haploids, suggesting that they adapt to lowered cyclin D level. Interestingly, however, haploid cells were more susceptible to cdk4/6 inhibition compared to diploids. Our finding indicates feasibility of selective growth suppression of haploid cells based on ploidy-linked gene regulation.

Requirements for diploid allogenic cell lines used for regenerative medicine

Requirements for diploid allogenic cell lines used for regenerative medicine

Toxic effects of copper sulfate on diploid and triploid fin cell lines in Misgurnus anguillicaudatus

The effects of different concentrations of copper sulfate on diploid and triploid fin cell lines (named DIMF and TRMF, respectively) in Misgurnus anguillicaudatus were studied. The LC50 of copper sulfate estimated by an MTT assay was 268.39 in DIMF cells, and 311.54 μmol/L in TRMF cells, respectively. Activity of superoxide dismutase (SOD) in DIMF cells gradually increased as the concentration of copper sulfate increased (up to 200 μmol/L), and then gradually decreased. SOD activity in triploid loach fin cells, as well as glutathione peroxidase (GSH-Px) and glutathione-S-transferase (GST) activity in both diploid and triploid cells, decreased as the concentration of copper sulfate increased, which suggested that excessive copper exposure at the concentrations tested in this study was detrimental to anti-oxidative capability. In general, SOD, GST and GSH-Px activity was higher in triploid fin cells than in diploid cells. DNA breaks were observed by comet assays after 24 h exposure to 400 and 800 μM copper; DNA percent in the comet's tail was lower in TRMF than in DIMF. Ultrastructurally, there were no significant differences in the organelles of both cells, although a higher number of vesicles were observed in TRMF cells after copper exposure. Pathological changes induced by copper sulfate were similar in DIMF and TRMF cells, and were indicative of cell necrosis. Results above suggested that excessive copper sulfate exposure would lead to antioxidant enzymes activity reduction, along with antioxidant defenses disruption and superoxide radicals increasing, and then to DNA damage, ultrastructural changes and necrosis features in DIMF and TRMF M. anguillicaudatus fin cells. Triploid cell lines had higher resistance to copper than their diploid counterparts especially at higher concentrations of copper due to larger cells and higher intracellular content of detoxification enzymes to resist the toxicity of heavy metals.

CDKN2A Depletion Causes Aneuploidy and Enhances Cell Proliferation in Non-Immortalized Normal Human Cells

Aneuploidy is a common feature of cancer cells and may contribute to cellular transformation and cancer development. In this study, we found that significant down-regulation of CDKN2A, CHEK2, CDCA8, TP53BP1, and CCNDBP1 led to chromosome imbalances in two diploid non-immortalized human cell lines; however, only CDKN2A inhibition enhanced cell proliferation and additionally up-regulated three cell cycle control genes: CDCA8, AURKA, and CCND. These results confirm that CDKN2A is a tumor suppressor gene driving human cancer development by inducing cell aneuploidy and cell cycle up-regulation.

Similarity in replication timing between polytene and diploid cells is associated with the organization of the Drosophila genome.

Morphologically, polytene chromosomes of Drosophila melanogaster consist of compact “black” bands alternating with less compact “grey” bands and interbands. We developed a comprehensive approach that combines cytological mapping data of FlyBase-annotated genes and novel tools for predicting cytogenetic features of chromosomes on the basis of their protein composition and determined the genomic coordinates for all black bands of polytene chromosome 2R. By a PCNA immunostaining assay, we obtained the replication timetable for all the bands mapped. The results allowed us to compare replication timing between polytene chromosomes in salivary glands and chromosomes from cultured diploid cell lines and to observe a substantial similarity in the global replication patterns at the band resolution level. In both kinds of chromosomes, the intervals between black bands correspond to early replication initiation zones. Black bands are depleted of replication initiation events and are characterized by a gradient of replication timing; therefore, the time of replication completion correlates with the band length. The bands are characterized by low gene density, contain predominantly tissue-specific genes, and are represented by silent chromatin types in various tissues. The borders of black bands correspond well to the borders of topological domains as well as to the borders of the zones showing H3K27me3, SUUR, and LAMIN enrichment. In conclusion, the characteristic pattern of polytene chromosomes reflects partitioning of the Drosophila genome into two global types of domains with contrasting properties. This partitioning is conserved in different tissues and determines replication timing in Drosophila.

Validation of a next generation sequencing-based preimplantation genetic screening assay for the calling of triploidy and uniparental isodisomy

We have previously validated the FAST-SeqS NGS-based PGS technology for the calling of whole chromosome and segmental aneuploidy (1,2). This technology captures sequence elements that cover ten to twenty thousand polymorphic sites. The relative representation of alleles at such sites can be used to call additional forms of chromosomal abnormality associated with negative reproductive outcomes, including triploidy, the presence of an extra set of chromosomes, and uniparental isodisomy (UPiD), the presence of two copies of the same chromosome from a single parent. To validate FAST-SeqS for the calling of triploidy and uniparental isodisomy. Samples from five triploid (69,XXY and 69,XXX), three UPiD (UPiD(7), UPiD(8), UPiD(1-22,X)) and eight diploid (47,XY,+9, 47,XY,+13, 47,XX,+18, 47,XY,+21, 47,XXY, 47,XYY, 46,XY, 46,XX) cell lines were run through FAST-SeqS. The resulting ploidy and UPiD calls were compared to the expected karyotypes to estimate analytical sensitivity and specificity. Next, to evaluate the dependence of triploidy and UPiD calling on cell count, cells from the lines noted above were run through FAST-SeqS at one, two, five or ten cells. Finally, to estimate the rate of triploidy and UPiD in blastocysts, we processed the FAST-SeqS data for 7365 embryos utilizing our validated analysis pipeline. All 76 triploid and 36 UPiD samples were called correctly, leading to triploidy and UPiD sensitivity estimates (95% confidence intervals) of 100% (95.8-100%) and 100% (98.4-100%), respectively. There were no false positive triploidy or UPiD calls for 210 known diploid samples, yielding triploidy and UPiD specificity estimates of 100% (98.2-100%) and 100% (98.2-100%), respectively. Across the two, five, and ten cell known triploid/UPiD samples (n=43, 53, 105), we observed zero false negative triploidy or UPiD calls. Similarly, no false positive triploidy or UPiD calls were made for the one, two, and five cell diploid samples (n=64, 132, 210). Amongst 7365 blastocyst samples, 69 were called triploid and 6 UPiD, yielding rate estimates of 0.94% (0.71-1.2%) and 0.08% (0.04-0.18%), respectively, values that are consistent with previous estimates (3-4). Of note, 33 of the triploid embryos were 69,XXX and therefore would not have been identifiable by an atypical sex chromosome copy number ratio. We have validated an enhanced version of the FAST-SeqS PGS assay for the accurate calling of triploidy and uniparental isodisomy, forms of chromosomal abnormality that are often not detected on other PGS platforms.

High Efficiency Gene Correction in Hematopoietic Cells by Donor-Template-Free CRISPR/Cas9 Genome Editing.

The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD)—a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs). Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.

Mosaic genome-wide maternal isodiploidy: an extreme form of imprinting disorder presenting as prenatal diagnostic challenge

BackgroundUniparental disomy of certain chromosomes are associated with a group of well-known genetic syndromes referred to as imprinting disorders. However, the extreme form of uniparental disomy affecting the whole genome is usually not compatible with life, with the exception of very rare cases of patients with mosaic genome-wide uniparental disomy reported in the literature.ResultsWe here report on a fetus with intrauterine growth retardation and malformations observed on prenatal ultrasound leading to invasive prenatal testing. By cytogenetic (conventional karyotyping), molecular cytogenetic (QF-PCR, FISH, array), and methylation (MS-MLPA) analyses of amniotic fluid, we detected mosaicism for one cell line with genome-wide maternal uniparental disomy and a second diploid cell line of biparental inheritance with trisomy X due to paternal isodisomy X. As expected for this constellation, we observed DNA methylation changes at all imprinted loci investigated.ConclusionsThis report adds new information on phenotypic outcome of mosaic genome-wide maternal uniparental disomy leading to an extreme form of multilocus imprinting disturbance. Moreover, the findings highlight the technical challenges of detecting these rare chromosome disorders prenatally.

Isolation and Structural Characterization of Echinocystic Acid Triterpenoid Saponins from the Australian Medicinal and Food Plant Acacia ligulata.

The Australian plant Acacia ligulata has a number of traditional food and medicinal uses by Australian Aboriginal people, although no bioactive compounds have previously been isolated from this species. Bioassay-guided fractionation of an ethanolic extract of the mature pods of A. ligulata led to the isolation of the two new echinocystic acid triterpenoid saponins, ligulatasides A (1) and B (2), which differ in the fine structure of their glycan substituents. Their structures were elucidated on the basis of 1D and 2D NMR, GC-MS, LC-MS/MS, and saccharide linkage analysis. These are the first isolated compounds from A. ligulata and the first fully elucidated structures of triterpenoid saponins from Acacia sensu stricto having echinocystic acid reported as the aglycone. Compounds 1 and 2 were evaluated for cytotoxic activity against a human melanoma cancer cell line (SK-MEL28) and a diploid fibroblast cell line (HFF), but showed only weak activity.

Identification of sites of 2\u2032-O-methylation vulnerability in human ribosomal RNAs by systematic mapping

Ribosomal RNA modifications are important in optimizing ribosome function. Sugar 2′-O-methylation performed by fibrillarin-associated box C/D antisense guide snoRNAs impacts all steps of translation, playing a role in disease etiology (cancer). As it renders adjacent phosphodiester bonds resistant to alkaline treatment, 2′-O-methylation can be monitored qualitatively and quantitatively by applying next-generation sequencing to fragments of randomly cleaved RNA. We remapped all sites of 2′-O-methylation in human rRNAs in two isogenic diploid cell lines, one producing and one not producing the antitumor protein p53. We identified sites naturally modified only partially (confirming the existence in cells of compositionally distinct ribosomes with potentially specialized functions) and sites whose 2′-O-methylation is sensitive to p53. We mapped sites particularly vulnerable to a reduced level of the methyltransferase fibrillarin. The remarkable fact that these are largely sites of natural hypomodification provides initial insights into the mechanism of partial RNA modification. Sites where methylation appeared vulnerable lie peripherally on the 3-D structure of the ribosomal subunits, whereas the numerous modifications present at the core of the subunits, where the functional centers lie, appeared robustly made. We suggest that vulnerable sites of 2′-O-methylation are highly likely to undergo specific regulation during normal and pathological processes.

The inner cell mass confirms deletion syndromes detected in trophectoderm biopsies

Deletion syndromes are defined as clinically recognizable genetic disorders characterized by a small chromosomal deletion, with a frequency of 0.5% in prenatal testing and 1 in 700 newborns. The cumulative effect of the imbalance on multiple independent disease genes within the deletion region determines the overall phenotype including, among others, developmental delay, intellectual disability and dysmorphic features. Many deletion syndromes have a corresponding duplication syndrome that is generally more rare and associated with a milder phenotype. Deletions and duplications are typically random events that occur during gametogenesis. The aim of this study was to evaluate the clinical reliability of diagnosing chromosomal deletions in blastocyst trophectoderm (TE) biopsies. Prospective blinded single-center study. A total of 26 blastocysts identified with small chromosome deletions, using the VeriSeq™ NGS platform (Illumina), were donated with patient consent (mean maternal age = 36.3 ±4.0years; mean paternal age = 38.6 ±6.1years). Each blastocyst was re-biopsied into three separate segments that were individually, blindly re-analyzed using the same VeriSeq™ NGS platform performed at the same genetic laboratory (n=96 samples). After un-blinding, all four segments were compiled together for a complete picture of every individual human blastocyst, including representatives of the inner cell mass (ICM) and TE. The overall incidence of partial aneuploidy following TE biopsy of 10,783 human blastocysts was observed at 1% (n=110). The blinded re-analysis of 26 blastocysts with small chromosome deletions, validated the reliability of the original deletion diagnosis in 24 (92.3%) of the embryos. The small chromosome deletion was observed throughout the entire embryo, including all ICM and TE segments, for 14 blastocysts (53.9%). The remaining blastocysts displayed evidence of potential mitotic cell division errors with either a diploid cell line in 1 TE segment (n=6; 23%), or the reverse small chromosome duplication in the re-biopsy ICM and TE segments (n=4; 15.4%). Examples of the clinically recognizable genetic disorders diagnosed in these 26 human blastocysts include chromosome 1q41-q42 deletion syndrome, chromosome 1p36 deletion syndrome, Cri-du-chat syndrome (end of chromosome 5p deletion) and chromosome 8q21.3-q22.1 deletion syndrome. This novel study validated the reliability of diagnosing chromosomal deletion syndromes in blastocyst biopsies. Our re-analysis results confirmed the original partial aneuploidy diagnosis to be present throughout the corresponding blastocyst with no preferential allocation to trophectoderm cells. These observed embryonic chromosomal deletions have been clearly associated with clinically recognizable genetic disorders.

Pushing the haystack aside for efficient gene targeting in human cells.

Gene targeting, i.e., the disruption of a specific gene via homologous recombination, is one of the key techniques to study gene function. However, researchers have faced the difficulty that, in human cells, the frequency of correct targeted integration is quite low compared to non-targeted (or random) integration. Adachi and colleagues investigated the factors that influence the ratio of targeted integration to non-targeted integration.

RIG-I and IL-6 are negative-feedback regulators of STING induced by double-stranded DNA

The stimulator of interferon genes (STING) protein has emerged as a critical signal transduction molecule in the innate immune response. Sustained activation of the STING signaling induced by cytosolic DNA has been considered to be the cause of a variety of autoimmune diseases characterized by uncontrolled inflammation. Therefore, it is important to understand the molecular basis of the regulation of STING signaling pathway. Here we demonstrate that the STING protein undergoes a proteasome-mediated degradation in human diploid cell (HDC) lines including MRC-5 following the transfection of double-stranded DNA (dsDNA). The degradation of STING is accompanied by the increased expression of both RIG-I and IL-6. Employing the RIG-I siRNA knockdown and an IL-6 neutralizing antibody greatly inhibits the degradation of STING induced by dsDNA. We further demonstrate that both IL-6 and RIG-I are downstream molecules of STING along the DNA sensor pathway. Therefore, STING degradation mediated by RIG-I and IL-6 may serve as a negative feedback mechanism to limit the uncontrolled innate immune response induced by dsDNA. We have further shown that RIG-I and IL-6 promote STING degradation by activating/dephosphorylating UNC-51-like kinase (ULK1). Interestingly, the STING protein is not significantly affected by dsDNA in non-HDC HEK293 cells. Our study thus has identified a novel signaling pathway for regulating STING in HDCs.

Abstract 3457: Stable aneuploid cells are more sensitive to TTK inhibition than chromosome instable cell lines

Abstract Inhibition of the spindle assembly checkpoint kinase TTK causes chromosome mis-segregation and tumor cell death. High levels of TTK correlate with chromosomal instability (CIN), which can lead to aneuploidy. To investigate the potential relationship of CIN and sensitivity to TTK inhibition, we performed CIN analysis in human cancer cell lines from different tumor tissue origins and with different relative sensitivity to the selective TTK inhibitor NTRC 0066-0 [1]. By time lapse microscopy we observed that treatment with TTK inhibitor resulted in overriding of the mitotic checkpoint, irrespective of cell line sensitivity. Aneuploid but chromosomal stable cell lines were more sensitive than cell lines that already displayed high levels of CIN. In sensitive cell lines, treatment with TTK inhibitor induced acute chromosome mis-segregration. On the contrary, in populations of cells that already showed high levels of CIN, there was only a small additional fraction of cells mis-segregating their chromosomes. The resistant cell lines were all hypo-triploid instable cell lines, which are thought to be derived from cells with a double diploid genome (tetraploid cells) that have lost certain chromosomes. Next, we studied the effect of NTRC 0066-0 on three cell lines evolved via tetraploidization of the colorectal adenocarcinoma cell line HCT 116 referred as tetraploids. The tetraploids have low levels of CIN and were recently shown to display low level multidrug resistance against various cytotoxic agents and several targeted drugs. Proliferation of the tetraploids was inhibited with the same potency as the diploid parental cell line by three TTK inhibitors. Parental and tetraploids were also equally sensitive to reversine, a compound that is often used as a TTK reference inhibitor but whose selectivity has been disputed. In surface plasmon resonance binding experiments we measured a 800 times more potent binding of reversine to TTK over Aurora B. In a comparative cancer cell panel profiling study with 122 different anti-cancer agents [2], reversine clustered together with TTK inhibitors, demonstrating that in cells reversine acts as a TTK inhibitor. Finally, we show that NTRC 0066-0 inhibits the proliferation of primary human patient-derived colorectal cancer organoids with potencies similar to that of immortalized cancer cell lines. In contrast, treatment with TTK inhibitor did not reduce the viability T cell acute lymphoblastic leukemia cell samples, which are non-proliferating cells and very sensitive to classic chemotherapeutic agents, such as daunorubicin. These data is consistent with the function of TTK as a spindle assembly checkpoint kinase that is only active in proliferating cells. Consequently, TTK inhibitor therapy is expected to spare non-dividing cells, whereas it may be used to target stable aneuploid tumors. [1] Maia et al. (2015) Annals of Oncology; [2] Uitdehaag et al. (2016) Molecular Cancer Therapeutics. Citation Format: Marion A.A. Libouban, Jeroen A.D.M. de Roos, Joost C.M. Uitdehaag, Nicole Willemsen-Seegers, Sara Mainardi, Jelle Dylus, Jos de Man, Bastiaan Tops, Jules P.P. Meijerink, Zuzana Storchová, Rogier C. Buijsman, René H. Medema, Guido J.R. Zaman. Stable aneuploid cells are more sensitive to TTK inhibition than chromosome instable cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3457. doi:10.1158/1538-7445.AM2017-3457

Heteroduplex cleavage assay for screening of probable zygosities resulting from CRISPR mutations in diploid single cell lines.

The most common gene editing methods, such as CRISPR, involve random repair of an induced double-stranded DNA break through the non-homologous end joining (NHEJ) repair pathway, resulting in small insertions/deletions. In diploid cells, these mutations can take on one of three zygosities: monoallelic, diallelic heterozygous, or diallelic homozygous. While many advances have been made in CRISPR delivery systems and gene editing efficiency, little work has been done to streamline detection of gene editing events. The only current method to determine the zygosity of an edited gene in a diploid organism is DNA sequencing, which is costly and time-consuming. Here, we describe the development of a T7 endonuclease I (T7EI)-based heteroduplex cleavage assay, along with statistical models relating the percentage of cleaved DNA to the zygosity of a mutation, that provides a rapid screening step prior to DNA sequencing. By isolating candidates likely to contain the desired zygosity for the edited gene, our screening method can decrease the number of clones requiring DNA sequencing.

Mislocalization of centromeric histone H3 variant CENP-A contributes to chromosomal instability (CIN) in human cells.

Chromosomal instability (CIN) is a hallmark of many cancers and a major contributor to tumorigenesis. Centromere and kinetochore associated proteins such as the evolutionarily conserved centromeric histone H3 variant CENP-A, associate with centromeric DNA for centromere function and chromosomal stability. Stringent regulation of cellular CENP-A levels prevents its mislocalization in yeast and flies to maintain genome stability. CENP-A overexpression and mislocalization are observed in several cancers and reported to be associated with increased invasiveness and poor prognosis. We examined whether there is a direct relationship between mislocalization of overexpressed CENP-A and CIN using HeLa and chromosomally stable diploid RPE1 cell lines as model systems. Our results show that mislocalization of overexpressed CENP-A to chromosome arms leads to chromosome congression defects, lagging chromosomes, micronuclei formation and a delay in mitotic exit. CENP-A overexpressing cells showed altered localization of centromere and kinetochore associated proteins such as CENP-C, CENP-T and Nuf2 leading to weakened native kinetochores as shown by reduced interkinetochore distance and CIN. Importantly, our results show that mislocalization of CENP-A to chromosome arms is one of the major contributors for CIN as depletion of histone chaperone DAXX prevents CENP-A mislocalization and rescues the reduced interkinetochore distance and CIN phenotype in CENP-A overexpressing cells. In summary, our results establish that CENP-A overexpression and mislocalization result in a CIN phenotype in human cells. This study provides insights into how overexpression of CENP-A may contribute to CIN in cancers and underscore the importance of understanding the pathways that prevent CENP-A mislocalization for genome stability.

Bone morphogenetic protein and Notch signalling crosstalk in poor-prognosis, mesenchymal-subtype colorectal cancer.

The functional role of bone morphogenetic protein (BMP) signalling in colorectal cancer (CRC) is poorly defined, with contradictory results in cancer cell line models reflecting the inherent difficulties of assessing a signalling pathway that is context‐dependent and subject to genetic constraints. By assessing the transcriptional response of a diploid human colonic epithelial cell line to BMP ligand stimulation, we generated a prognostic BMP signalling signature, which was applied to multiple CRC datasets to investigate BMP heterogeneity across CRC molecular subtypes. We linked BMP and Notch signalling pathway activity and function in human colonic epithelial cells, and normal and neoplastic tissue. BMP induced Notch through a γ‐secretase‐independent interaction, regulated by the SMAD proteins. In homeostasis, BMP/Notch co‐localization was restricted to cells at the top of the intestinal crypt, with more widespread interaction in some human CRC samples. BMP signalling was downregulated in the majority of CRCs, but was conserved specifically in mesenchymal‐subtype tumours, where it interacts with Notch to induce an epithelial–mesenchymal transition (EMT) phenotype. In intestinal homeostasis, BMP–Notch pathway crosstalk is restricted to differentiating cells through stringent pathway segregation. Conserved BMP activity and loss of signalling stringency in mesenchymal‐subtype tumours promotes a synergistic BMP–Notch interaction, and this correlates with poor patient prognosis. BMP signalling heterogeneity across CRC subtypes and cell lines can account for previous experimental contradictions. Crosstalk between the BMP and Notch pathways will render mesenchymal‐subtype CRC insensitive to γ‐secretase inhibition unless BMP activation is concomitantly addressed. © 2017 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.