Diploid Cell Line Research Articles

Comparing the cytotoxic potential of <i>Withania somnifera</i> water and methanol extracts

The plant Withania somnifera (Linn.) (Solanacea) is a well-known herbal medicine used in many parts of the world. It has anti-inflammatory, antioxidant, and antitumor as well as neural protective properties. It seems as if the two most active withanolide components, namely withaferin A and withanolide D, found in methanol (MeOH) extracts, are responsible for the anti-inflammatory and antioxidant properties of the plant. The current research evaluated and compared the cytotoxic potential of water and methanol extracts of W. somnifera using a combined crystal violet MTT and Neutral Red assay. MRC-5 cells, a human embryonic lung-derived diploid fibroblast cell line, were the cells of choice. We found that the three lowest concentrations (0.007, 0.042, 0.250 microg/ml) of the plant material extracted in double distilled H(2)O and MeOH do not differ significantly in any of the assays. We therefore suggest that low concentrations of MeOH extracts (up to 0.250 microg/ml plant material) do not cause cell damage to the MRC-5 cells, however, higher levels should be avoided as cell viability and cell numbers are negatively influenced.

Development and characterization of three new diploid cell lines from Labeo rohita (Ham.)

Development of cell lines from fish for identifying the pathogenesis of viral diseases and for vaccine production against viral and bacterial diseases is imperative where they are of commercial importance. Three new diploid fish cell lines (RF, RH, and RSB) were developed from fin, heart, and swim bladder of an Indian major carp, Labeo rohita, commonly called Rohu. All the cell lines were optimally maintained at 28 degrees C in Leibovitz-15 medium supplemented with 10% FBS. The propagation of RH and RSB cells was serum dependent, with a low plating efficiency (<16%), whereas RF cells showed 20% efficiency. The cytogenetic analysis revealed a diploid count of 50 chromosomes. The cells of RF and RSB were found to be epithelial, where as the cells of RH were mostly fibroblastic. The viability of the RF, RH, and RSB cell lines was 75, 70 and 72%, respectively after 6 months of storage in liquid nitrogen. The origin of the cell lines was confirmed by the amplification of 496 and 655 bp fragments of 16S rRNA and Cytochrome Oxidase Subunit I (COI) of mtDNA. The new cell lines would facilitate viral disease diagnosis and genomic studies.

2-Aryl-4,5,6,7-tetrahydro-1,3-benzothiazol-7-ols as a class of antitumor agents selectively active in securin −/− cells

A series of 2-(4-aminophenyl)-4,5,6,7-tetrahydro-1,3-benzothiazol-7-ols have been developed as antitumor agents that showed high selectivity against aneuploid cell lines (vs diploid cell lines). Structure–activity relationship studies showed that a hydroxymethyl group at the 2-position of the phenyl ring increased potency and selectivity. A pyrrolidinyl group at the 4-position of the phenyl ring was comparable to a dimethylamino group. The corresponding 5-aza analogs, 2-(4-aminophenyl)-4,5,6,7-tetrahydro[1,3]thiazolo[4,5- c]pyridin-7-ols, retained potency and high level of selectivity against aneuploid cell growth (vs diploid cells). These 5-aza compounds exhibited higher water solubility and higher metabolic stability than the corresponding carba analogs. Compound 19 showed the highest potency against MCF-7 and MDA-MB-361 lines and was selected for further evaluation.

Advantages and difficulties in culturing human pluripotent stem cells in growth factor-defined serum-free medium

Advantages and difficulties in culturing human pluripotent stem cells in growth factor-defined serum-free medium

Abstract 4391: 4-Hydroxy-2-nonenal causes tetraploidy through a macrophage-induced bystander effect triggered by a commensal bacterium

Abstract Tetraploidy can be an intermediate condition for cells undergoing tranformation from diploidy to aneuploidy. We previously showed that Enterococcus faecalis, a human intestinal commensal, generated tetraploidy and aneuploidy in colonic epithelial cells via a macrophage-induced bystander effect. To explore potential mediators for this effect, supernatants from E. faecalis-infected macrophages were analyzed by HPLC coupled to a coulometric detection system. Significantly increased concentrations of 4-hydroxy-2-nonenal (4-HNE), a breakdown product of polyunsaturated fatty acids, were detected in macrophage supernatants. This system was used to purify macrophage-generated 4-HNE for further experiments. To investigate aneugenic effects of 4-HNE, a near-diploid human colon cancer cell line (HCT116) and non-transformed diploid mouse colonic epithelial cell line (YAMC) were exposed to 4-HNE and ploidy analyzed by FACS. For HCT116 cells the percent tetraploidy significantly increased following exposure to 4-HNE at 2.5 µM compared to untreated controls (9.90% ± 0.09 vs. 0.21% ± 0.08, P &amp;lt; 0.001). In comparison, the percent tetraploidy was increased to 0.68% ± 0.10 for YAMC cells exposed to 1 µM 4-HNE compared to 0.12% ± 0.06 for untreated controls (P &amp;lt; 0.001). This showed that 4-HNE generated tetraploidy in both transformed and non-transformed cells. To address potential mechanisms by which 4-HNE produced tetraploidy, HCT116 cells exposed to 4-HNE were stained with anti-α/β tubulin antibody and microtubules analyzed by confocal microscopy. We found cells exposed to 4-HNE had degraded microtubules and abnormal mitotic patterns, implicating the induction of tetraploidy through inhibition of microtubule assembly. Moreover, for HCT116 and YAMC cells, 4-HNE caused a G2/M cell cycle arrest with increased γH2AX foci suggesting DNA double-strand breaks. Next, we silenced mGSTα4-4, a key enzyme in the detoxification of 4-HNE, by constitutively expressing RNAi in cells. FACS analysis showed that γH2AX positivity remarkably increased in YAMC cells with silenced mGSTα4-4 that were exposed to E. faecalis-infected macrophages compared to non-infected macrophages. No changes were seen in wildtype YAMC cells. Similarly, γH2AX foci increased in mGSTα4-4-silenced YAMC cells exposed to 4-HNE compared to the wildtype control (41.8% ± 1.3 vs. 24.6% ± 6.5, P = 0.01). Finally, percent tetraploidy also significantly increased for mGSTα4-4-silenced YAMC cells exposed to 4-HNE, supporting the hypothesis that 4-HNE can specifically generate tetraploidy. In sum, E. faecalis-infected macrophages produce 4-HNE that, in turn, induces tetraploidy, DNA double-strand breaks and G2/M arrest in colonic epithelial cells. Inactivation of mGSTα4-4, a detoxifying enzyme for 4-HNE, exacerbated these effects. We propose 4-HNE as a mediator for the E. faecalis-infected macrophage bystander effect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4391.

ETV6/RUNX1 abrogates mitotic checkpoint function and targets its key player MAD2L1

Approximately 25% of childhood B-cell precursor acute lymphoblastic leukemia have an ETV6/RUNX1 (E/R) gene fusion that results from a t(12;21). This genetic subgroup of leukemia is associated with near-triploidy, near-tetraploidy, and trisomy 21 as rather specific types of secondary changes. Here, we show that, unlike various controls, E/R-expressing Ba/F3 clones acquire a tetraploid karyotype on prolonged culture, corroborating the assumption that E/R may attenuate the mitotic checkpoint (MC). Consistent with this notion, E/R-expressing diploid murine and human cell lines have decreased proportions of cells with 4N DNA content and a lower mitotic index when treated with spindle toxins. Moreover, both RUNX1 and E/R regulate mitotic arrest-deficient 2 L1 (MAD2L1), an essential MC component, by binding to promoter-inherent RUNX1 sites, which results in down-regulation of MAD2L1 mRNA and protein in E/R-expressing cells. Forced expression of E/R also abolishes RUNX1-induced reporter activation, whereas E/R with a mutant DNA-binding site leads to only minor effects. Our data link for the first time E/R, MC, and MAD2L1 and provide new insights into the function of the E/R fusion gene product. Although tetraploidy is an almost exclusive feature of E/R-positive leukemias, its rarity within this particular subgroup implies that further yet unknown factors are required for its manifestation.

TheIn VitroandIn VivoAntitumor Activity of Adenovirus-Mediated Interleukin-24 Expression for Laryngocarcinoma

Interleukin-24 (IL-24)/melanoma differentiation associated gene-7 (mda-7) as a novel tumor-suppressor gene has potent antitumor activities in a broad spectrum of human cancers through the activation of various signaling pathways. However, the suppressive effect of adenovirus-mediated IL-24 (Ad-IL-24) expression on human laryngeal cancers is still elusive. In this study, we explored the therapeutic effect of Ad-IL-24 on human laryngeal cancers in vitro and in vivo in an athymic nude mouse model, using a Hep-2 human laryngocarcinoma cell line, and a WI-38 human diploid cell line served as a normal cell control. We demonstrated that Ad-IL-24 induced significant growth inhibition and apoptosis, upregulated the expression of P21, P27, and Bax, downregulated Bcl-2 expression, and activated caspase-3 in Hep-2 laryngeal tumor cells, while it exerted no direct effect on the in vitro proliferation of WI-38 normal diploid cells. Moreover, intratumoral injections of Ad-IL-24 in nude mice bearing Hep-2 tumors significantly suppressed the laryngeal xengrafted tumor growth and reduced microvessel density (MVD) and VEGF expression in tumors. This retarded tumor growth in vitro and in vivo elicited by Ad-IL-24 was closely associated with the upregulation of proliferation-related molecules P21 and P27, decrease in the ratio of anti- to proapoptotic molecules Bcl-2/Bax, followed by the activation of caspase-3, leading to apoptosis via intrinsic apoptotic pathways, and the reduced expression of proangiogenic factor VEGF involved in the inhibition of tumor angiogenesis. Thus, our results indicate that the potent, selective killing activity of Ad-IL-24 in laryngeal cancer cells, but not in normal cells, makes this vector a potential candidate for laryngeal cancer gene therapy.

Abstract A79: Genome-wide RNAi screen for tetraploid-specific lethality in cancer cells

Abstract Cancer cells are often genetically unstable, resulting in aneuploid genomes. The consequences of aneuploidy are poorly understood, but have been suggested to play roles in tumor aggressiveness, chemotherapy resistance, and metastasis. One important route to an aneuploid genome may be through doubling of the genome, leading to unstable tetraploid cells. Many cell division errors are ultimately manifest as a failure of cytokinesis. Especially in cells defective in p53 function, tetraploid cells proliferate, but generate both high rates of whole chromosome aneuploidy and defects in the maintenance of chromosome structural integrity. Our laboratory has demonstrated that, in p53-primary cells, tetraploidy promotes tumor development and the resulting tumors display markedly abnormal genomes (Fujiwara T. et al. Nature 2005, 437: 1043). It is now known that several human cancer-mutations — e.g. Rb loss or loss of the adenomatous polyposis coli tumor suppressor — also predispose cytokinesis failure and tetraploidy. These findings highlight a need to understand in detail the physiological changes that accompany tetraploidy. To gain a comprehensive understanding of physiological alterations associated with tetraploidy in cancer cells, we have established tetraploid cell lines derived from a near diploid human colorectal cancer cell line HCT116. Using these paired tetraploid and diploid cell lines, we have performed a genome-wide RNAi screen to identify genes whose knock down selectively affect the viability of either diploids or tetraploid cells. We are currently validating the result of our ploidy-specific lethality screen and we would like to update our progress at this conference. Citation Information: Cancer Res 2009;69(23 Suppl):A79.

Synthesis and antiproliferative evaluation of 6-arylindeno[1,2- c]quinoline derivatives

A number of 6-arylindeno[1,2- c]quinoline derivatives were synthesized and evaluated for their antiproliferative activities against the growth of five cancer cell lines including human hepatocelluar carcinoma (Hep G2, Hep 3B and Hep2.2.1), non-small cell lung cancer (A549 and H1299), and normal diploid embryonic lung cell line (MRC-5). The preliminary results indicated that 9-(3-(dimethylamino)propoxy)-6-(4-(3-(dimethylamino)propoxy)phenyl)-2-fluoro-11 H-indeno[1,2- c]quinolin-11-one ( 14c) was the most potent with GI 50 values of 0.61, 0.67, 0.59, and 0.72 μM against the growth of Hep G2, Hep 3B, Hep 2.2.1, and H1299 cells, respectively. Results have also shown that 2,9-bis(3-(dimethylamino)propoxy)-6-(4-(3-(dimethylamino)propoxy)phenyl)-11 H-indeno[1,2- c]quinolin-11-one ( 17), which exhibited GI 50 of 0.60 and 0.68 μM against the growth of Hep G2 and A549, respectively, was more active than the positive topotecan and irinotecan. Compound 17 was less toxic than topotecan against the growth of normal cell (MRC-5) and therefore, was selected for further evaluation. Results indicated that compound 17 induce cell cycle arrest in G2/M phase, DNA fragmentation, and disrupt the microtubule network in A549 cells. The apoptotic induction may through the cleavage of PARP.

Parthenogenic activation of discarded in vitro matured, vitrified and rewarmed human oocytes for stem cell production

OBJECTIVE: Oocyte parthenogenic activation (PA) is a potential source of embryonic stem cells. We performed PA on discarded human oocytes that were in vitro matured, vitrified and warmed, to assess the developmental potential of parthenotes for stem cell production. DESIGN: Prospective analysis of parthenogenically activated human oocytes. MATERIALS AND METHODS: Discarded oocytes were obtained from consenting patients. Immature oocytes were cultured in 5% Human Serum Albumin in Quinn's Cleavage Media for ∼20 hours to mature in vitro. All mature oocytes were vitrified and warmed using either the Medicult or Irvine Scientific systems (Medicult Vitrification Cooling and Warming kits of Medicult, Denmark; Irvine Scientific Vitrification Freeze and Warm kits of Irvine Scientific, Santa Ana, CA). Oocytes surviving ∼2 hours post-warming were used for PA. Oocytes were exposed to 5μM calcium ionomysin (CI) then puromycin dihydrochloride (puro, 10μg/ml) or 6-dimethylaminopurine (6-DMAP, 1mM). Activated oocytes were cultured in 10% Synthetic Serum Substitute in Quinn's Cleavage Media. Chi-square analysis was performed. RESULTS: See table.Table 1Vitrification SystemSurvival Rate (%)Activation MethodActivation Rate (%)Medicult48/50 (90)CI/puro16/24 (66.7)CI/6-DMAP10/24 (41.7)Irvine Scientific25/34 (73.5)CI/puro8/15 (53.3)CI/6-DMAP6/10 (60)Total73/84 (86.9)All40/73 (54.8) Open table in a new tab CONCLUSIONS: Oocyte PA has the potential to create HLA-matched stem cell lines. Using discarded oocytes that were matured in vitro, vitrified and warmed, we successfully demonstrated that such oocytes could undergo PA and develop to Day 3 (40/73, 54.8%) . Further studies to obtain Day 5 parthenotes for genetic and epigenetic analysis are ongoing. It is our hope to ultimately create diploid autologous stem cell lines for future experimentation and possible stem cell transfers.

Establishment and characterization of an ovarian cell line of the silkworm, Bombyx mori

A cell line BmN-SWU1 was established from the ovarian tissues of 3-day-old fourth instar Bombyx mori larvae of the 21-872nlw variety by performing primary cultures in Grace's medium supplemented with 20% fetal bovine serum (FBS). The cell line primarily consisted of short spindle cells and round cells. The frequency of cells with chromosome number 2 n = 56 was 80.5%; therefore, the cell line was considered to be a diploid cell line. The population-doubling time (PDT) at 45th passage line was 57.7 h. This cell line was susceptible to the B. mori nuclear polyhedrovirus (BmNPV), and the median tissue culture infective dose (TCID 50) at a cell density of 10 5 cells/ml was 16.3 OBs/ml. The transient expression efficiency of the green fluorescent protein (GFP) gene in this cell line was 54.8%. We used the BmN-SWU1 cell line to select and establish a GFP transgenic cell line.

Sesquiterpenoids from Teucrium ramosissimum

An antiplasmodial bioguided investigation of the EtOAc extract of the aerial parts of Teucrium ramosissimum led to isolation and identification of three sesquiterpenoids, teucmosin, 4α-hydroxy-homalomenol C, 1β,4β,7α-trihydroxy-8,9-eudesmene and two trinorsesquiterpenoids, 4β-hydroxy-11,12,13-trinor-5-eudesmen-1,7-dione and 1β,4β-dihydroxy-11,12,13-trinor-8,9-eudesmen-7-one together with five known sesquiterpenoids, oplopanone, homalomenol C, oxo- T-cadinol, 1β,4β,6β-trihydroxyeudesmane, 1β,4β,7α-trihydroxyeudesmane and four flavonoids, 5-hydroxy-7,4′-dimethoxyflavone, salvigenin, genkwanin and cirsimaritin. The structures and the relative stereochemistry were elucidated by extensive spectroscopic studies including 1D and 2D NMR and mass spectrometry (MS). Homalomenol C, 4β-hydroxy-11,12,13-trinor-5-eudesmen-1,7-dione, oxo- T-cadinol and 1β,4β,6β-trihydroxyeudesmane displayed a significant in vitro antiplasmodial activity against Plasmodium falciparum with IC 50 values ranging from 1.2 to 5.0 μg/ml. Furthermore, no cytotoxicity was observed upon the human diploid lung cell line MRC-5 for these compounds.

The Probability to Initiate X Chromosome Inactivation Is Determined by the X to Autosomal Ratio and X Chromosome Specific Allelic Properties

BackgroundIn female mammalian cells, random X chromosome inactivation (XCI) equalizes the dosage of X-encoded gene products to that in male cells. XCI is a stochastic process, in which each X chromosome has a probability to be inactivated. To obtain more insight in the factors setting up this probability, we studied the role of the X to autosome (X∶A) ratio in initiation of XCI, and have used the experimental data in a computer simulation model to study the cellular population dynamics of XCI.Methodology/Principal FindingsTo obtain more insight in the role of the X∶A ratio in initiation of XCI, we generated triploid mouse ES cells by fusion of haploid round spermatids with diploid female and male ES cells. These fusion experiments resulted in only XXY triploid ES cells. XYY and XXX ES lines were absent, suggesting cell death related either to insufficient X-chromosomal gene dosage (XYY) or to inheritance of an epigenetically modified X chromosome (XXX). Analysis of active (Xa) and inactive (Xi) X chromosomes in the obtained triploid XXY lines indicated that the initiation frequency of XCI is low, resulting in a mixed population of XaXiY and XaXaY cells, in which the XaXiY cells have a small proliferative advantage. This result, and findings on XCI in diploid and tetraploid ES cell lines with different X∶A ratios, provides evidence that the X∶A ratio determines the probability for a given X chromosome to be inactivated. Furthermore, we found that the kinetics of the XCI process can be simulated using a probability for an X chromosome to be inactivated that is proportional to the X∶A ratio. These simulation studies re-emphasize our hypothesis that the probability is a function of the concentration of an X-encoded activator of XCI, and of X chromosome specific allelic properties determining the threshold for this activator.ConclusionsThe present findings reveal that the probability for an X chromosome to be inactivated is proportional to the X∶A ratio. This finding supports the presence of an X-encoded activator of the XCI process.

Low Concentrations of Hydrogen Peroxide Induce Altered Physiology of Human Diploid Fibroblasts

The objective of this study was to determine the physiological effects of low‐dose hydrogen peroxide (H2O2) treatments on the cellular physiology of GM00701, GM01354, GM01385, MRC‐5, and MRC‐9 human diploid fibroblast cell lines. A number of published reports have shown short‐term treatments of fibroblasts with H2O2 concentrations of 100 uM to 200 uM result in apoptosis. However, our preliminary studies show that 5.0 uM to 25.0 uM concentrations of H2O2 are capable of inducing apoptosis in the cell lines that were studied. One‐hour treatments of fibroblasts with these H2O2 concentrations were shown to result in statistically significant decreases in cell number. The relative cell numbers are measured 24 hours after treatment by Alamar Blue cytotoxicity assay. We also used flow cytometry to analyze the effects of H2O2 treatment on cell cycle progression in a time‐dependent manner 12 and 24 hours post‐treatment. We find a 1.5‐ to 3‐fold increase in sub G1 cells 24 hours after treatment when compared to untreated cells. The results are cell line specific. To confirm these findings, studies are currently underway to assess the effects of H2O2 treatment on intracellular reduced GSH concentrations and Poly (ADP‐ribose) polymerase (PARP) cleavage. We will assess the effects of sub‐lethal doses of H2O2 on the metabolism of low density lipoprotein (LDL) and oxidized low density lipoprotein (ox‐LDL) by flow cytometry. We will also assess the effects of sub‐lethal doses of H2O2 on cytokine‐induced IL‐6 and MCP‐1 secretion by ELISA. From our studies, we conclude that low levels of oxidative stress can have significant effects on the physiology of human fibroblasts.

Differential toxic effects of methyl tertiary butyl ether and tert-butanol on rat fibroblasts in vitro

Methyl tertiary butyl ether (MTBE) is the most widely used motor vehicle fuel oxygenate since it reduces harmful emissions due to gasoline combustion. However, the significant increase in its use in recent years has raised new questions related to its potential toxicity. In fact, although available data are somehow conflicting, there is evidence that MTBE is a toxic substance that may have harmful effects on both animals and humans and an unresolved problem is the role played by MTBE metabolites, especially tertiary butyl alcohol (TBA), in determining toxic effects due to MTBE exposure. In this study, the toxic effects of MTBE have been analyzed on a normal diploid rat fibroblast cell line (Rat-1) and compared to the effects of TBA. The results obtained suggest that both MTBE and TBA inhibit cell growth in vitro but with different mechanisms in terms of effects on the cell cycle progression and on the modulation of cell cycle regulatory proteins. In fact, MTBE caused an accumulation of cells in the S-phase of the cell cycle, whereas TBA caused an accumulation in the G0/G1-phase with different effects on the expression of cyclin D1, p27Kip1, and p53. Moreover, both MTBE and TBA were also shown to induce DNA damage, as assessed in terms of oxidative DNA damage and nuclear DNA fragmentation, that appeared to be susceptible of repair by the cell DNA-repair machinery. In conclusion, these findings suggest that both MTBE and TBA can exert, by acting through different molecular mechanisms, important biological effects on fibroblasts in vitro. Further studies are warranted to shed light on the mechanisms responsible for the observed effects and on their potential significance for the in-vivo exposure.

Asynaptic meiosis in fission yeast?

Meiotic prophase has been studied by both light and electron microscopy in diploid cell lines (h+/h-) of Schizosaccharomyces pombe. One wild-type and one mutant strain were used, the latter (mei4-B2) being blocked after premeiotic DNA synthesis and after commitment to meiosis. Meiosis and sporulation were induced in liquid culture by a shift to a sporulation medium devoid of a nitrogen source. The time course was determined from light-microscopic preparations; most cells passing through meiotic prophase between 5 and 8 h after the shift. In samples from that interval, ca. 20% of the nuclei were elongate in shape, containing strands of chromatin in rough alignement with the long axis of the nucleus. This stage was found to accumulate in the mutant. A bundle of microtubules, just outside of the spindle plaque, apparently stabilized the elongated shape of the nuclei. Linear electron-dense structures were observed to populate these nuclei. They resembled unpaired lateral elements of synaptonemal complexes in other organisms. Yet, at no times did we observe a tripartite complex or any other sign of chromosomal pairing in our material.

Introduction: Uveal melanoma

Introduction: Uveal melanoma

Human Glyceraldehyde-3-phosphate Dehydrogenase Plays a Direct Role in Reactivating Oxidized Forms of the DNA Repair Enzyme APE1

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has diverse biological functions including its nuclear translocation in response to oxidative stress. We show that GAPDH physically associates with APE1, an essential enzyme involved in the repair of abasic sites in damaged DNA, as well as in the redox regulation of several transcription factors. This interaction allows GAPDH to convert the oxidized species of APE1 to the reduced form, thereby reactivating its endonuclease activity to cleave abasic sites. The GAPDH variants C152G and C156G retain the ability to interact with but are unable to reactivate APE1, implicating these cysteines in catalyzing the reduction of APE1. Interestingly, GAPDH-small interfering RNA knockdown sensitized the cells to methyl methane sulfonate and bleomycin, which generate lesions that are repaired by APE1, but showed normal sensitivity to 254-nm UV. Moreover, the GAPDH knockdown cells exhibited an increased level of spontaneous abasic sites in the genomic DNA as a result of diminished APE1 endonuclease activity. Thus, the nuclear translocation of GAPDH during oxidative stress constitutes a protective mechanism to safeguard the genome by preventing structural inactivation of APE1.

Xist RNA is confined to the nuclear territory of the silenced X chromosome throughout the cell cycle.

In mammalian female cells, one X chromosome is inactivated to prevent a dose difference in the expression of X-encoded proteins between males and females. Xist RNA, required for X chromosome inactivation, is transcribed from the future inactivated X chromosome (Xi), where it spreads in cis, to initiate silencing. We have analyzed Xist RNA transcription and localization throughout the cell cycle. It was found that Xist transcription is constant and that the mature RNA remains attached to the Xi throughout mitosis. Diploid and tetraploid cell lines with an MS2-tagged Xist gene were used to investigate spreading of Xist. Most XXXX(MS2) tetraploid mouse embryonic stem (ES) cells inactivate the X(MS2) chromosome and one other X chromosome. Analysis of cells with two Xi's indicates that Xist RNA is retained by the Xi of its origin and does not spread in trans. Also, in XX(MS2) diploid mouse ES cells with an autosomal Xist transgene, there is no trans exchange of Xist RNA from the Xi to the autosome. We propose that Xist RNA does not dissociate from the Xi of its origin, which precludes a model of diffusion-mediated trans spreading of Xist RNA.

Human Sgo1 downregulation leads to chromosomal instability in colorectal cancer

Background and aims:Chromosomal instability (CIN) is recognised as a hallmark of cancer and is caused by a spindle assembly checkpoint disorder or chromosome mis-segregation during mitosis. Although the recent identification...