scholarly journals Mapping the phenotypic repertoire of the cytoplasmic 2-Cys peroxiredoxin – thioredoxin system. 1. Understanding commonalities and differences among cell types

2017 ◽  
Author(s):  
Gianluca Selvaggio ◽  
Pedro M. B. M. Coelho ◽  
Armindo Salvador
Keyword(s):  
Cell Lines ◽  
Thioredoxin Reductase ◽  
Cell Types ◽  
Cell Physiology ◽  
Related Factor ◽  
Similar Response ◽  
Redox Biology ◽  
Analytical Approximations ◽  
Thioredoxin System ◽  
Reduction Capacity

AbstractThe system (PTTRS) formed by typical 2-Cys peroxiredoxins (Prx), thioredoxin (Trx), Trx reductase (TrxR), and sulfiredoxin (Srx) is central in antioxidant protection and redox signaling in the cytoplasm of eukaryotic cells. Understanding how the PTTRS integrates these functions requires tracing phenotypes to molecular properties, which is non-trivial. Here we analyze this problem based on a model that captures the PTTRS’ conserved features. We have mapped the conditions that generate each distinct response to H2O2 supply rates (νsup), and estimated the parameters for thirteen human cell types and for Saccharomyces cerevisiae. The resulting composition-to-phenotype map yielded the following experimentally testable predictions. The PTTRS permits many distinct responses including ultra-sensitivity and hysteresis. However, nearly all tumor cell lines showed a similar response characterized by limited Trx-S- depletion and a substantial but self-limited gradual accumulation of hyperoxidized Prx at high νsup. This similarity ensues from strong correlations between the TrxR, Srx and Prx activities over cell lines, which contribute to maintain the Prx-SS reduction capacity in slight excess over the maximal steady state Prx-SS production. In turn, in erythrocytes, hepatocytes and HepG2 cells high νsup depletes Trx-S- and oxidizes Prx mainly to Prx-SS. In all nucleated human cells the Prx-SS reduction capacity defined a threshold separating two different regimes. At sub-threshold νsup cytoplasmic H2O2 is determined by Prx, nM-range and spatially localized, whereas at supra-threshold νsup it is determined by much less active alternative sinks and μM-range throughout the cytoplasm. The yeast shows a distinct response where the Prx Tsa1 accumulates in sulfenate form at high νsup. This is mainly due to an exceptional stability of Tsa1’s sulfenate.The implications of these findings for thiol redox regulation and cell physiology are discussed. All estimates were thoroughly documented and provided, together with analytical approximations for system properties, as a resource for quantitative redox biology.AbbreviationsASK1, apoptosis signal-regulating kinase 1; Cat, catalase; GSH, glutathione; GPx1, glutathione peroxidase 1; Grx, glutaredoxin; KEAP1, Kelch-like ECH-associated protein 1; NRF2, nuclear factor erythroid 2-related factor 2; Prx, typical 2-Cys peroxiredoxin; PTTRS, peroxiredoxin / thioredoxin / thioredoxin reductase system; Srx, sulfiredoxin; Trx, thioredoxin; TrxR, thioredoxin reductase.

scholarly journals Systematic identification of a nuclear receptor-enriched predictive signature for erastin-induced ferroptosis

2020 ◽  
Author(s):  
Ok-Seon Kwon ◽  
Eun-Ji Kwon ◽  
Hyeon-Joon Kong ◽  
Jeong-Yoon Choi ◽  
Yun-Jeong Kim ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Nuclear Receptor ◽  
Cancer Cell ◽  
Cell Types ◽  
Cancer Cell Lines ◽  
Glutathione Depletion ◽  
Related Factor ◽  
Sensitivity Testing ◽  
Aryl Hydrocarbon

AbstractErastin, which has been initially identified as a synthetic lethal compound against cancer expressing an RAS oncogene, inhibits cystine/glutamate antiporters and causes ferroptic cell death in various cell types, including therapy-resistant mesenchymal cancer cells. However, despite recent emerging evidence for the mechanisms underlying ferroptosis, molecular biomarkers associated with erastin-dependent ferroptosis have not yet been identified. In the present study, we employed isogenic lung cancer cell models with therapy-resistant mesenchymal properties to show that a redox imbalance leads to glutathione depletion and ferroptotic cell death. Subsequent gene expression analysis of pan-cancer cell lines revealed that the activity of transcription factors, including nuclear factor erythroid 2-related factor 2 (NRF2) and aryl hydrocarbon receptor (AhR), serve as important markers of erastin resistance. Based on the integrated expression of genes in the nuclear receptor meta-pathway (NRM), we constructed an NRM model and validated its robustness using an independent pharmacogenomics dataset. The NRM model was further evaluated by employing it in the sensitivity testing of nine cancer cell lines for which erastin sensitivities had not yet been undetermined. Our pharmacogenomics approach has the potential to pave the way for the efficient classification of patients for therapeutic intervention using erastin or erastin analogs.

scholarly journals Human Immunodeficiency Viruses Pseudotyped with SARS-CoV-2 Spike Proteins Infect a Broad Spectrum of Human Cell Lines through Multiple Entry Mechanisms

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 953
Author(s):  
Chuan Xu ◽  
Annie Wang ◽  
Ke Geng ◽  
William Honnen ◽  
Xuening Wang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Viral Entry ◽  
Broad Spectrum ◽  
A549 Cells ◽  
Cell Types ◽  
Pseudotyped Virus ◽  
Angiotensin Converting Enzyme 2 ◽  
Human Immunodeficiency Viruses ◽  
Tyrosine Protein Kinase ◽  
Overexpressing Cell

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), enters cells through attachment to the human angiotensin converting enzyme 2 (hACE2) via the receptor-binding domain (RBD) in the surface/spike (S) protein. Several pseudotyped viruses expressing SARS-CoV-2 S proteins are available, but many of these can only infect hACE2-overexpressing cell lines. Here, we report the use of a simple, two-plasmid, pseudotyped virus system comprising a SARS-CoV-2 spike-expressing plasmid and an HIV vector with or without vpr to investigate the SARS-CoV-2 entry event in various cell lines. When an HIV vector without vpr was used, pseudotyped SARS-CoV-2 viruses produced in the presence of fetal bovine serum (FBS) were able to infect only engineered hACE2-overexpressing cell lines, whereas viruses produced under serum-free conditions were able to infect a broader range of cells, including cells without hACE2 overexpression. When an HIV vector containing vpr was used, pseudotyped viruses were able to infect a broad spectrum of cell types regardless of whether viruses were produced in the presence or absence of FBS. Infection sensitivities of various cell types did not correlate with mRNA abundance of hACE2, TMPRSS2, or TMPRSS4. Pseudotyped SARS-CoV-2 viruses and replication-competent SARS-CoV-2 virus were equally sensitive to neutralization by an anti-spike RBD antibody in cells with high abundance of hACE2. However, the anti-spike RBD antibody did not block pseudotyped viral entry into cell lines with low abundance of hACE2. We further found that CD147 was involved in viral entry in A549 cells with low abundance of hACE2. Thus, our assay is useful for drug and antibody screening as well as for investigating cellular receptors, including hACE2, CD147, and tyrosine-protein kinase receptor UFO (AXL), for the SARS-CoV-2 entry event in various cell lines.

scholarly journals Infection, dissemination, and transmission efficiencies of Zika virus in Aedes aegypti after serial passage in mosquito or mammalian cell lines or alternating passage in both cell types

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Lourdes G. Talavera-Aguilar ◽  
Reyes A. Murrieta ◽  
Sungmin Kiem ◽  
Rosa C. Cetina-Trejo ◽  
Carlos M. Baak-Baak ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Cell Lines ◽  
Zika Virus ◽  
Cell Types ◽  
Mosquito Cell ◽  
Cell Passage ◽  
Genetic Changes ◽  
Synonymous Substitutions ◽  
Vertebrate Cell

Abstract Background Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) with an urban transmission cycle that primarily involves humans and Aedes aegypti. Evidence suggests that the evolution of some arboviruses is constrained by their dependency on alternating between disparate (vertebrate and invertebrate) hosts. The goals of this study are to compare the genetic changes that occur in ZIKV after serial passaging in mosquito or vertebrate cell lines or alternate passaging in both cell types and to compare the replication, dissemination, and transmission efficiencies of the cell culture-derived viruses in Ae. aegypti. Methods An isolate of ZIKV originally acquired from a febrile patient in Yucatan, Mexico, was serially passaged six times in African green monkey kidney (Vero) cells or Aedes albopictus (C6/36) cells or both cell types by alternating passage. A colony of Ae. aegypti from Yucatan was established, and mosquitoes were challenged with the cell-adapted viruses. Midguts, Malpighian tubules, ovaries, salivary glands, wings/legs and saliva were collected at various times after challenge and tested for evidence of virus infection. Results Genome sequencing revealed the presence of two non-synonymous substitutions in the premembrane and NS1 regions of the mosquito cell-adapted virus and two non-synonymous substitutions in the capsid and NS2A regions of both the vertebrate cell-adapted and alternate-passaged viruses. Additional genetic changes were identified by intrahost variant frequency analysis. Virus maintained by continuous C6/36 cell passage was significantly more infectious in Ae. aegypti than viruses maintained by alternating passage and consecutive Vero cell passage. Conclusions Mosquito cell-adapted ZIKV displayed greater in vivo fitness in Ae. aegypti compared to the other viruses, indicating that obligate cycling between disparate hosts carries a fitness cost. These data increase our understanding of the factors that drive ZIKV adaptation and evolution and underscore the important need to consider the in vivo passage histories of flaviviruses to be evaluated in vector competence studies. Graphic abstract "Image missing"

scholarly journals The plasminogen system and cell surfaces: evidence for plasminogen and urokinase receptors on the same cell type.

1986 ◽  
Vol 103 (6) ◽  
pp. 2411-2420 ◽  
Author(s):  
E F Plow ◽  
D E Freaney ◽  
J Plescia ◽  
L A Miles
Keyword(s):  
Cell Line ◽  
Plasminogen Activator ◽  
Cell Lines ◽  
Specific Binding ◽  
High Capacity ◽  
Fetal Lung ◽  
Fibroblast Cell ◽  
Cell Types ◽  
Cell Surfaces ◽  
Catalytically Active

The capacity of cells to interact with the plasminogen activator, urokinase, and the zymogen, plasminogen, was assessed using the promyeloid leukemic U937 cell line and the diploid fetal lung GM1380 fibroblast cell line. Urokinase bound to both cell lines in a time-dependent, specific, and saturable manner (Kd = 0.8-2.0 nM). An active catalytic site was not required for urokinase binding to the cells, and 55,000-mol-wt urokinase was selectively recognized. Plasminogen also bound to the two cell lines in a specific and saturable manner. This interaction occurred with a Kd of 0.8-0.9 microM and was of very high capacity (1.6-3.1 X 10(7) molecules bound/cell). The interaction of plasminogen with both cell types was partially sensitive to trypsinization of the cells and required an unoccupied high affinity lysine-binding site in the ligand. When plasminogen was added to the GM1380 cells, a line with high intrinsic plasminogen activator activity, the bound ligand was comprised of both plasminogen and plasmin. Urokinase, in catalytically active or inactive form, enhanced plasminogen binding to the two cell lines by 1.4-3.3-fold. Plasmin was the predominant form of the bound ligand when active urokinase was added, and preformed plasmin can also bind directly to the cells. Plasmin on the cell surface was also protected from its primary inhibitor, alpha 2-antiplasmin. These results indicate that the two cell lines possess specific binding sites for plasminogen and urokinase, and a family of widely distributed cellular receptors for these components may be considered. Endogenous or exogenous plasminogen activators can generate plasmin on cell surfaces, and such activation may provide a mechanism for arming cell surfaces with the broad proteolytic activity of this enzyme.

scholarly journals Human Immunodeficiency Virus Type 1 Replication in the Absence of Integrase-Mediated DNA Recombination: Definition of Permissive and Nonpermissive T-Cell Lines

2001 ◽  
Vol 75 (17) ◽  
pp. 7944-7955 ◽  
Author(s):  
Noriko Nakajima ◽  
Richard Lu ◽  
Alan Engelman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Dna Recombination ◽  
Cell Types ◽  
Class Ii ◽  
Class I ◽  
Immunodeficiency Virus ◽  
T Cell Lines ◽  
Hiv 1

ABSTRACT Functional retroviral integrase protein is thought to be essential for productive viral replication. Yet, previous studies differed on the extent to which integrase mutant viruses expressed human immunodeficiency virus type 1 (HIV-1) genes from unintegrated DNA. Although one reason for this difference was that class II integrase mutations pleiotropically affected the viral life cycle, another reason apparently depended on the identity of the infected cell. Here, we analyzed integrase mutant viral infectivities in a variety of cell types. Single-round infectivity of class I integration-specific mutant HIV-1 ranged from <0.03 to 0.3% of that of the wild type (WT) across four different T-cell lines. Based on this approximately 10-fold influence of cell type on mutant gene expression, we examined class I and class II mutant replication kinetics in seven different cell lines and two primary cell types. Unexpectedly, some cell lines supported productive class I mutant viral replication under conditions that restricted class II mutant growth. Cells were defined as permissive, semipermissive, or nonpermissive based on their ability to support the continual passage of class I integration-defective HIV-1. Mutant infectivity in semipermissive and permissive cells as quantified by 50% tissue culture infectious doses, however, was only 0.0006 to 0.005% of that of WT. Since the frequencies of mutant DNA recombination in these lines ranged from 0.023 to <0.093% of the WT, we conclude that productive replication in the absence of integrase function most likely required the illegitimate integration of HIV-1 into host chromosomes by cellular DNA recombination enzymes.

scholarly journals Antioxidant Activities and Selenogene Transcription in the European Sea Bass (Dicentrarchus labrax) Liver Depend, in a Non-linear Manner, on the Se/Hg Molar Ratio of the Feeds

2021 ◽  
Author(s):  
Marinelle Espino ◽  
Harkaitz Eguiraun ◽  
Oihane Diaz de Cerio ◽  
José Antonio Carrero ◽  
Nestor Etxebarria ◽  
...  
Keyword(s):  
Antioxidant Activities ◽  
Dicentrarchus Labrax ◽  
Thioredoxin Reductase ◽  
Sea Bass ◽  
Molar Ratio ◽  
Antioxidant Systems ◽  
European Sea Bass ◽  
Thioredoxin System ◽  
Non Linear ◽  
Linear Manner

AbstractFeeding 3.9 and 6.7 mg Hg/kg (Se/Hg molar ratios of 0.8 and 0.4, respectively) for 14 days negatively affected Dicentrarchus labrax growth and total DNTB- and thioredoxin-reductase (TrxR) activities and the transcription of four redox genes (txn1, gpx1, txnrd3, and txnrd2) in the liver, but a diet with 0.5 mg Hg/kg (Se/Hg molar ratio 6.6) slightly increased both reductase activities and the transcription of txn1, gpx1, and txnrd2. Feeding 6.7 mg Hg/kg for 53 days downregulated the genes of the thioredoxin system (txn1, txnrd3, and txnrd2) but upregulated gpx1, confirming the previously proposed complementarity among the antioxidant systems. Substitution of 20% of the feed by thawed white fish (hake) slightly counteracted the negative effects of Hg. The effects were not statistically significant and were dependent, in a non-linear manner, on the Se/Hg molar ratio of the feed but not on its Hg concentration. These results stress the need to consider the Se/Hg molar ratio of the feed/food when evaluating the toxicity of Hg.

Cellular Moloney murine sarcoma (c-mos) sequences are hypermethylated and transcriptionally silent in normal and transformed rodent cells

1982 ◽  
Vol 2 (1) ◽  
pp. 42-51
Author(s):  
S Gattoni ◽  
P Kirschmeier ◽  
I B Weinstein ◽  
J Escobedo ◽  
D Dina
Keyword(s):  
Cell Lines ◽  
Blot Hybridization ◽  
Cell Types ◽  
Single Copy ◽  
Transformed Cell Line ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Blotting Technique ◽  
Moloney Sarcoma Virus ◽  
Murine Sarcoma

Moloney murine sarcoma virus carries an oncogenic sequence (v-mos) which is homologous to a single copy gene (c-mos) present in the normal cells of several vertebrate species. Because of the possible significance of c-mos sequences in normal development and malignant transformation induced by physical or chemical agents, we have examined the state of integration, methylation, and transcriptional activity of c-mos sequences in a variety of normal rodent tissues, normal cell lines, or cell lines transformed by radiation or chemical carcinogens. DNA-DNA hybridization, utilizing the Southern blotting technique and a plasmid-derived DNA probe representing the v-mos sequence, gave no evidence for rearrangements of the c-mos sequence in the DNAs obtained from these diverse cell types. Parallel studies employing the restriction enzyme isoschizomers HpaII and MspI indicated that in all of these cell types the c-mos sequences were heavily methylated. In addition, analysis of cellular RNAs by blot hybridization with the v-mos probe failed to detect evidence of transcription of the c-mos sequences in any of these cell types. This was in contrast to a Moloney sarcoma virus-transformed cell line in which we found that the integrated v-mos sequence was both undermethylated and extensively transcribed. Thus, it would appear that c-mos sequences do not play a role in the transformation of rodent cells by chemical or physical agents, although the possible role of other endogenous onc sequences remains to be determined.

Downregulation of Sulfiredoxin (Srx) Suppressed Cell Proliferation Migration and Invasion Through Inhibiting Extracellular Signal-Regulated Kinase/Nrf2 Pathway in Hepatocellular Carcinoma

2021 ◽  
Vol 11 (11) ◽  
pp. 2137-2145
Author(s):  
Xuejuan Zhu ◽  
Danqian Lu
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Colony Formation ◽  
Migration And Invasion ◽  
Related Factor ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Nrf2 Signaling Pathway

Background: Sulfiredoxin (Srx) has been identified to play important roles in the development of various cancers. However, the precise effects and underlying mechanism of Srx on the progression of HCC are far from being fully understood. Materials and Methods: The abundances of Srx in THLE-2 cell and HCC cell lines were determined by western blot and RT-qPCR. Next, SK-Hep-1 cells were transfected with shRNA-Srx or shRNA-NC and treated with TBHQ (an extracellular signal-regulated kinase (ERK) activator) for functional experiments. Then, CCK8 and colony formation assays were used to determine cell proliferation and clone-forming abilities in vitro. Cell migration and invasion were assessed via wound healing and transwell assays. The expression of MMP2, MMP9 and key members in ERK/nuclear factor E2 related factor (Nrf2) signaling pathway was detected by performing western blot analysis. Results: We reported evidence that Srx was frequently up-regulated in HCC cell lines. Srx interference constrained cell proliferation, colony formation rate, migration and invasion of SK-Hep-1 cells. Moreover, mechanistic investigations indicated that Srx interference significantly inhibited the activation of ERK/Nrf2 signaling pathway, and ERK activator TBHQ can reverse the functions of Srx interference in SK-Hep-1 cells. Conclusion: Overall, Downregulation of Srx might impede HCC progression by suppressing ERK/Nrf2 signaling pathway. Findings in the current study reported the functional involvement and molecular mechanism of Srx in HCC, suggesting that Srx might have a potential therapeutic value in HCC treatment.

Invasive behaviour of glioblastoma cell lines is associated with altered organisation of the cadherin-catenin adhesion system

2002 ◽  
Vol 115 (16) ◽  
pp. 3331-3340 ◽  
Author(s):  
Carla Perego ◽  
Cristina Vanoni ◽  
Silvia Massari ◽  
Andrea Raimondi ◽  
Sandra Pola ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Lines ◽  
Protein A ◽  
Cell Types ◽  
Glioblastoma Cell ◽  
Migration Assay ◽  
Adhesion System ◽  
Pdz Protein ◽  
Glioblastoma Cell Lines ◽  
Cell Cell

As little is known about the role of cadherin-mediated cell-cell adhesion in astrocytes and its alteration in migrating and invasive glioblastomas, we investigated its molecular composition and organisation in primary cultured astrocytes and the T98G and U373MG glioblastoma cell lines. Biochemical and morphological analysis indicated that all three cell types express all of the structural components of the adhesion system, including the LIN-7 PDZ protein,a novel component involved in the organisation of the junctional domain in epithelia and neurons. However, only the astrocytes and T98G cells generated and maintained mature adhesive junctional domains to which LIN-7 was recruited. Alterations in the junctional domain of U373MG cells were associated with higher motility in a poly-L-lysine migration assay. When the T98G cells were cultured on Matrigel matrix, they acquired invasive properties but, despite unchanged cadherin adhesion system protein levels, the invasive T98G cell-cell contacts failed to accumulate LIN-7 and failed to mature. These results identify the LIN-7 PDZ protein as a marker of cell adhesion maturity and cell invasion and indicate that instability and disorganisation of cadherin-mediated junctions rather than reduced expression of cadherin-catenin system components are required to promote migration and invasiveness in glioblastoma cell lines.

Deficiency in intercellular communication in two established renal epithelial cell lines (LLC-PK1 and MDCK)

1984 ◽  
Vol 66 (1) ◽  
pp. 81-93
Author(s):  
F.V. Sepulveda ◽  
J.D. Pearson
Keyword(s):  
Epithelial Cell ◽  
Cell Lines ◽  
Mdck Cells ◽  
Cell Communication ◽  
Intercellular Junctions ◽  
Cell Types ◽  
Renal Epithelial Cell ◽  
Aortic Endothelial Cells ◽  
Epithelial Cell Lines ◽  
Autoradiographic Analysis

We have studied the cell-to-cell passage of uridine nucleotides in two renal epithelial cell lines (LLC-PK1 and MDCK) and in porcine aortic endothelial cells (PAE). All three cell types incorporated tritiated uridine. After a 3 h incubation the radioactivity was predominantly in the form of acid-soluble compounds, mainly UTP. Prelabelled LLC-PK1 or MDCK cells were unable to transfer radioactivity to added adjacent, non-labelled cells, whereas PAE cells readily formed communicating intercellular junctions, as judged by autoradiographic analysis after a 3 h co-culture period. Cell-to-cell communication in either of the renal cell lines was not promoted by treatment with dibutyryl cyclic AMP and methylisobutylxanthine. Radioactivity incorporated into the acid-insoluble pool was not available for intercellular transfer, as assessed in experiments in which cells were prelabelled 24 h before co-culture.

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