scholarly journals Uncoordinated centrosome cycle underlies the instability of non-diploid somatic cells in mammals

2018 ◽  
Vol 217 (7) ◽  
pp. 2463-2483 ◽  
Author(s):  
Kan Yaguchi ◽  
Takahiro Yamamoto ◽  
Ryo Matsui ◽  
Yuki Tsukada ◽  
Atsuko Shibanuma ◽  
...  
Keyword(s):  
Dna Replication ◽  
Human Cell ◽  
Ploidy Level ◽  
Somatic Cells ◽  
Cell Types ◽  
Centrosome Duplication ◽  
Human Cell Lines ◽  
Scaling Properties ◽  
Gradual Loss ◽  
Ploidy Changes

In animals, somatic cells are usually diploid and are unstable when haploid for unknown reasons. In this study, by comparing isogenic human cell lines with different ploidies, we found frequent centrosome loss specifically in the haploid state, which profoundly contributed to haploid instability through subsequent mitotic defects. We also found that the efficiency of centriole licensing and duplication changes proportionally to ploidy level, whereas that of DNA replication stays constant. This caused gradual loss or frequent overduplication of centrioles in haploid and tetraploid cells, respectively. Centriole licensing efficiency seemed to be modulated by astral microtubules, whose development scaled with ploidy level, and artificial enhancement of aster formation in haploid cells restored centriole licensing efficiency to diploid levels. The ploidy–centrosome link was observed in different mammalian cell types. We propose that incompatibility between the centrosome duplication and DNA replication cycles arising from different scaling properties of these bioprocesses upon ploidy changes underlies the instability of non-diploid somatic cells in mammals.

scholarly journals Uncoordinated centrosome duplication cycle underlies the instability of non-diploid states in mammalian somatic cells

2017 ◽  
Author(s):  
Kan Yaguchi ◽  
Ryo Matsui ◽  
Takahiro Yamamoto ◽  
Yuki Tsukada ◽  
Atsuko Shibanuma ◽  
...  
Keyword(s):  
Dna Replication ◽  
Ploidy Level ◽  
Somatic Cells ◽  
Centrosome Duplication ◽  
Scaling Properties ◽  
Gradual Loss ◽  
Monopolar Spindle ◽  
Ploidy Changes ◽  
Duplication Cycle ◽  
Parthenogenetic Mouse Embryos

AbstractIn animals, somatic cells are usually diploid and are unstable when haploid for unknown reasons. In this study, by comparing isogenic human cell lines with different ploidies, we found frequent centrosome loss specifically in the haploid state, which profoundly contributed to haploid instability through monopolar spindle formation and subsequent mitotic defects. We also found that efficiency of centriole licensing and duplication, but not that of DNA replication, changes proportionally to ploidy level, causing gradual loss or frequent overduplication of centrioles in haploid and tetraploid cells, respectively. Centriole licensing efficiency seemed to be modulated by astral microtubules, whose development scaled with ploidy level, and artificial enhancement of aster formation in haploid cells restored centriole licensing efficiency to diploid levels. Haploid-specific centrosome loss was also observed in parthenogenetic mouse embryos. We propose that incompatibility between the centrosome duplication and DNA replication cycles arising from different scaling properties of these bioprocesses upon ploidy changes, underlies the instability of non-diploid somatic cells in mammals.SummaryYaguchi et al. show that a delay or acceleration of centriole licensing compromises the control of centrosome number in haploid or tetraploid human cells, respectively, suggesting a cellular basis of the instability of non-diploid somatic cells in mammals.

scholarly journals Analysis of Mitochondrial DNA Polymorphisms in the Human Cell Lines HepaRG and SJCRH30

2019 ◽  
Vol 20 (13) ◽  
pp. 3245 ◽  
Author(s):  
Matthew J. Young ◽  
Anitha D. Jayaprakash ◽  
Carolyn K. J. Young
Keyword(s):  
Mitochondrial Dna ◽  
Cell Lines ◽  
Human Cell ◽  
Cell Types ◽  
Dna Polymorphisms ◽  
Reference Sequence ◽  
Sequencing Analysis ◽  
Human Cell Lines ◽  
Mtdna Sequences ◽  
Mtdna Variants

The mitochondrial DNA (mtDNA) sequences of two commonly used human cell lines, HepaRG and SJCRH30, were determined. HepaRG originates from a liver tumor obtained from a patient with hepatocarcinoma and hepatitis C while SJCRH30 originates from a rhabdomyosarcoma patient tumor. In comparison to the revised Cambridge Reference Sequence, HepaRG and SJCRH30 mtDNA each contain 14 nucleotide variations. In addition to an insertion of a cytosine at position 315 (315insC), the mtDNA sequences from both cell types share six common polymorphisms. Heteroplasmic variants were identified in both cell types and included the identification of the 315insC mtDNA variant at 42 and 75% heteroplasmy in HepaRG and SJCRH30, respectively. Additionally, a novel heteroplasmic G13633A substitution in the HepaRG ND5 gene was detected at 33%. Previously reported cancer-associated mtDNA variants T195C and T16519C were identified in SJCRH30, both at homoplasmy (100%), while HepaRG mtDNA harbors a known prostate cancer-associated T6253C substitution at near homoplasmy, 95%. Based on our sequencing analysis, HepaRG mtDNA is predicted to lie within haplogroup branch H15a1 while SJCRH30 mtDNA is predicted to localize to H27c. The catalog of polymorphisms and heteroplasmy reported here should prove useful for future investigations of mtDNA maintenance in HepaRG and SJCRH30 cell lines.

scholarly journals HBO1 (KAT7) Does Not Have an Essential Role in Cell Proliferation, DNA Replication, or Histone 4 Acetylation in Human Cells

2019 ◽  
Vol 40 (4) ◽  
Author(s):  
Andrew J. Kueh ◽  
Samantha Eccles ◽  
Leonie Tang ◽  
Alexandra L. Garnham ◽  
Rose E. May ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Adhesion ◽  
Dna Replication ◽  
Cell Lines ◽  
Human Cell ◽  
Human Cells ◽  
Human Cell Lines ◽  
Hela Cell Lines ◽  
Mouse Tissues ◽  
Sirna Knockdown

ABSTRACT HBO1 (MYST2/KAT7) is essential for histone 3 lysine 14 acetylation (H3K14ac) but is dispensable for H4 acetylation and DNA replication in mouse tissues. In contrast, previous studies using small interfering RNA (siRNA) knockdown in human cell lines have suggested that HBO1 is essential for DNA replication. To determine if HBO1 has distinctly different roles in immortalized human cell lines and normal mouse cells, we performed siRNA knockdown of HBO1. In addition, we used CRISPR/Cas9 to generate 293T, MCF7, and HeLa cell lines lacking HBO1. Using both techniques, we show that HBO1 is essential for all H3K14ac in human cells and is unlikely to have a direct effect on H4 acetylation and only has minor effects on cell proliferation. Surprisingly, the loss of HBO1 and H3K14ac in HeLa cells led to the secondary loss of almost all H4 acetylation after 4 weeks. Thus, HBO1 is dispensable for DNA replication and cell proliferation in immortalized human cells. However, while cell proliferation proceeded without HBO1 and H3K14ac, HBO1 gene deletion led to profound changes in cell adhesion, particularly in 293T cells. Consistent with this phenotype, the loss of HBO1 in both 293T and HeLa principally affected genes mediating cell adhesion, with comparatively minor effects on other cellular processes.

scholarly journals SARS-CoV-2 disrupts proximal elements in the JAK-STAT pathway

2021 ◽  
Author(s):  
Da-Yuan Chen ◽  
Nazimuddin Khan ◽  
Brianna J. Close ◽  
Raghuveera K. Goel ◽  
Benjamin Blum ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Type I Interferon ◽  
Cell Types ◽  
Janus Kinase ◽  
Type I ◽  
Human Cell Lines ◽  
Interferon Signaling ◽  
Interferon Receptor ◽  
Stat Pathway

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we generated a panel of phenotypically diverse, SARS-CoV-2-infectable human cell lines representing different body organs and performed longitudinal survey of cellular proteins and pathways broadly affected by the virus. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cells and primary-like cardiomyocytes, and found that SARS-CoV-2 targeted the proximal pathway components, including Janus kinase 1 (JAK1), tyrosine kinase 2 (Tyk2), and the interferon receptor subunit 1 (IFNAR1), resulting in cellular desensitization to type I IFN. Detailed mechanistic investigation of IFNAR1 showed that the protein underwent ubiquitination upon SARS-CoV-2 infection. Furthermore, chemical Inhibition of JAK kinases enhanced infection of stem cell-derived cultures, indicating that the virus benefits from inhibiting the JAK-STAT pathway. These findings suggest that the suppression of interferon signaling is a mechanism widely used by the virus to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19. IMPORTANCE SARS-CoV-2 can infect various organs in the human body, but the molecular interface between the virus and these organs remains unexplored. In this study, we generated a panel of highly infectable human cell lines originating from various body organs and employed these cells to identify cellular processes commonly or distinctly disrupted by SARS-CoV-2 in different cell types. One among the universally impaired processes was interferon signaling. Systematic analysis of this pathway in diverse culture systems showed that SARS-CoV-2 targets the proximal JAK-STAT pathway components, destabilizes the type I interferon receptor though ubiquitination, and consequently renders the infected cells resistant to type I interferon. These findings illuminate how SARS-CoV-2 can continue to propagate in different tissues even in the presence of a disseminated innate immune response.

scholarly journals Uncoupling of DNA replication and centrosome duplication cycles is a primary cause of haploid instability in mammalian somatic cells

2020 ◽  
Author(s):  
Koya Yoshizawa ◽  
Kan Yaguchi ◽  
Ryota Uehara
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Dna Replication ◽  
Genome Instability ◽  
Somatic Cells ◽  
S Phase ◽  
Centrosome Duplication ◽  
Potential Contribution ◽  
Mitotic Control ◽  
The Stability

AbstractMammalian haploid somatic cells are unstable and prone to diploidize, but the cause of haploid instability remains largely unknown. Previously, we found that mammalian haploid somatic cells suffer chronic centrosome loss stemming from the uncoupling of DNA replication and centrosome duplication cycles. However, the lack of methodology to restore the coupling between DNA replication and centrosome duplication has precluded us from investigating the potential contribution of the haploidy-linked centrosome loss to haploid instability. In this study, we developed an experimental method that allows the re-coupling of DNA and centrosome cycles through the chronic extension of the G1/S phase without compromising cell proliferation using thymidine treatment/release cycles. Chronic extension of G1/S restored normal mitotic centrosome number and mitotic control, substantially improving the stability of the haploid state in HAP1 cells. Stabilization of the haploid state was compromised when cdk2 was inhibited during the extended G1/S, or when early G1 was chronically extended instead of G1/S, showing that the coupling of DNA and centrosome cycles rather than a general extension of the cell cycle is required for haploid stability. Our data indicate the chronic centriole loss arising from the uncoupling of centrosome and DNA cycles as a direct cause of genome instability in haploid somatic cells, and also demonstrate the feasibility of modulation of haploid stability through artificial coordination between DNA and centrosome cycles in mammalian somatic cells.

scholarly journals Enhanced Cellular Uptake of H-Chain Human Ferritin Containing Gold Nanoparticles

Pharmaceutics ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 1966
Author(s):  
Italo Moglia ◽  
Margarita Santiago ◽  
Simon Guerrero ◽  
Mónica Soler ◽  
Alvaro Olivera-Nappa ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Cell Lines ◽  
Cellular Uptake ◽  
Human Cell ◽  
Animal Experiments ◽  
Cell Types ◽  
Biological Properties ◽  
Human Cell Lines ◽  
Theranostic Agents

Gold nanoparticles (AuNP) capped with biocompatible layers have functional optical, chemical, and biological properties as theranostic agents in biomedicine. The ferritin protein containing in situ synthesized AuNPs has been successfully used as an effective and completely biocompatible nanocarrier for AuNPs in human cell lines and animal experiments in vivo. Ferritin can be uptaken by different cell types through receptor-mediated endocytosis. Despite these advantages, few efforts have been made to evaluate the toxicity and cellular internalization of AuNP-containing ferritin nanocages. In this work, we study the potential of human heavy-chain (H) and light-chain (L) ferritin homopolymers as nanoreactors to synthesize AuNPs and their cytotoxicity and cellular uptake in different cell lines. The results show very low toxicity of ferritin-encapsulated AuNPs on different human cell lines and demonstrate that efficient cellular ferritin uptake depends on the specific H or L protein chains forming the ferritin protein cage and the presence or absence of metallic cargo. Cargo-devoid apoferritin is poorly internalized in all cell lines, and the highest ferritin uptake was achieved with AuNP-loaded H-ferritin homopolymers in transferrin-receptor-rich cell lines, showing more than seven times more uptake than apoferritin.

scholarly journals Exploring Chromosomal Structural Heterogeneity Across Multiple Cell Lines

2020 ◽  
Author(s):  
Ryan R. Cheng ◽  
Vinicius Contessoto ◽  
Erez Lieberman Aiden ◽  
Peter G. Wolynes ◽  
Michele Di Pierro ◽  
...  
Keyword(s):  
Cell Lines ◽  
Conformational Changes ◽  
Human Cell ◽  
Conformational Transition ◽  
Cell Types ◽  
Structural Heterogeneity ◽  
Human Cell Lines ◽  
Active Chromatin ◽  
State Process ◽  
Structural Ensembles

AbstractWe study the structural ensembles of human chromosomes across different cell types. Using computer simulations, we generate cell-specific 3D chromosomal structures and compare them to recently published chromatin structures obtained through microscopy. We demonstrate using a combination of machine learning and polymer physics simulations that epigenetic information can be used to predict the structural ensembles of multiple human cell lines. The chromosomal structures obtained in silico are quantitatively consistent with those obtained through microscopy as well as DNA-DNA proximity ligation assays. Theory predicts that chromosome structures are fluid and can only be described by an ensemble, which is consistent with the observation that chromosomes exhibit no unique fold. Nevertheless, our analysis of both structures from simulation and microscopy reveals that short segments of chromatin make transitions between a closed conformation and an open dumbbell conformation. This conformational transition appears to be consistent with a two-state process with an effective free energy cost of about four times the effective information theoretic temperature. Finally, we study the conformational changes associated with the switching of genomic compartments observed in human cell lines. Genetically identical but epigenetically distinct cell types appear to rearrange their respective structural ensembles to expose segments of transcriptionally active chromatin, belonging to the A genomic compartment, towards the surface of the chromosome, while inactive segments, belonging to the B compartment, move to the interior. The formation of genomic compartments resembles hydrophobic collapse in protein folding, with the aggregation of denser and predominantly inactive chromatin driving the positioning of active chromatin toward the surface of individual chromosomal territories.

scholarly journals Two subunits of human ORC are dispensable for DNA replication and proliferation

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Etsuko Shibata ◽  
Manjari Kiran ◽  
Yoshiyuki Shibata ◽  
Samarendra Singh ◽  
Shashi Kiran ◽  
...  
Keyword(s):  
Dna Replication ◽  
Cell Lines ◽  
Human Cell ◽  
Origin Recognition Complex ◽  
Human Cell Lines ◽  
Atpase Subunit ◽  
Origins Of Replication ◽  
A Subunit ◽  
Dependent Mechanism ◽  
Origin Recognition

The six-subunit Origin Recognition Complex (ORC) is believed to be an essential eukaryotic ATPase that binds to origins of replication as a ring-shaped heterohexamer to load MCM2-7 and initiate DNA replication. We have discovered that human cell lines in culture proliferate with intact chromosomal origins of replication after disruption of both alleles of ORC2 or of the ATPase subunit, ORC1. The ORC1 or ORC2-depleted cells replicate with decreased chromatin loading of MCM2-7 and become critically dependent on another ATPase, CDC6, for survival and DNA replication. Thus, either the ORC ring lacking a subunit, even its ATPase subunit, can load enough MCM2-7 in partnership with CDC6 to initiate DNA replication, or cells have an ORC-independent, CDC6-dependent mechanism to load MCM2-7 on origins of replication

scholarly journals Expression of extracellular superoxide dismutase by human cell lines

1990 ◽  
Vol 266 (1) ◽  
pp. 213-219 ◽  
Author(s):  
S L Marklund
Keyword(s):  
Superoxide Dismutase ◽  
Cell Lines ◽  
Human Cell ◽  
Heparan Sulphate ◽  
Cell Types ◽  
Tissue Cell ◽  
Extracellular Superoxide Dismutase ◽  
Human Cell Lines ◽  
Growing Cell ◽  
Dependent Cell

Extracellular superoxide dismutase (EC-SOD) is the major SOD isoenzyme in extracellular fluids, but occurs also in tissues. The sites and characteristics of the synthesis of the enzyme are unknown. The occurrence of EC-SOD in cultures of a large panel of human cell lines was assayed by means of an e.l.i.s.a. Unlike the situation for the intracellular isoenzymes CuZn-SOD and Mn-SOD, expression of EC-SOD occurs in only a few cell types. None of the ten investigated suspension-growing cell lines produced EC-SOD. Among normal diploid anchorage-dependent cell lines, expression was found in all 25 investigated fibroblast cell lines, in the two glia-cell lines, but not in six endothelial-cell lines, two epithelial-cell lines or in two amnion-derived lines. Among neoplastic anchorage-dependent cell lines expression was found in 13 out of 29. EC-SOD was secreted into the culture medium by cell lines expressing the enzyme. The rate of EC-SOD synthesis varied by nearly 100-fold among the fibroblast lines and remained essentially constant in the individual lines during long-term culture. In the nine investigated cases, the secreted EC-SOD was of the high-heparin-affinity C type. It is suggested that tissue EC-SOD is secreted by a few well-dispersed cell types, such as fibroblasts and glia cells, to diffuse subsequently around and reversibly bind to heparan sulphate proteoglycan ligands in the glycocalyx of the surface of most tissue cell types and in the interstitial matrix.

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