scholarly journals Inactivation of N-Acetylglucosaminyltransferase I and α1,3-Fucosyltransferase Genes in Nicotiana tabacum BY-2 Cells Results in Glycoproteins With Highly Homogeneous, High-Mannose N-Glycans

2021 ◽  
Vol 12 ◽  
Author(s):  
Xavier Herman ◽  
Johann Far ◽  
Adeline Courtoy ◽  
Laurent Bouhon ◽  
Loïc Quinton ◽  
...  
Keyword(s):  
Nicotiana Tabacum ◽  
Cell Lines ◽  
Wild Type ◽  
Suspension Cells ◽  
Protein Activity ◽  
Recombinant Glycoproteins ◽  
Bright Yellow ◽  
High Mannose ◽  
Glycan Heterogeneity ◽  
High Degree

Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells are among the most commonly used plant cell lines for producing biopharmaceutical glycoproteins. Recombinant glycoproteins are usually produced with a mix of high-mannose and complex N-glycans. However, N-glycan heterogeneity is a concern for the production of therapeutic or vaccine glycoproteins because it can alter protein activity and might lead to batch-to-batch variability. In this report, a BY-2 cell line producing glycoproteins devoid of complex N-glycans was obtained using CRISPR/Cas9 edition of two N-acetylglucosaminyltransferase I (GnTI) genes, whose activity is a prerequisite for the formation of all complex N-glycans. The suppression of complex N-glycans in the GnTI-knocked out (KO) cell lines was assessed by Western blotting. Lack of β1,2-xylose residues confirmed the abolition of GnTI activity. Unexpectedly, α1,3-fucose residues were still detected albeit dramatically reduced as compared with wild-type cells. To suppress the remaining α1,3-fucose residues, a second genome editing targeted both GnTI and α1,3-fucosyltransferase (FucT) genes. No β1,2-xylose nor α1,3-fucose residues were detected on the glycoproteins produced by the GnTI/FucT-KO cell lines. Absence of complex N-glycans on secreted glycoproteins of GnTI-KO and GnTI/FucT-KO cell lines was confirmed by mass spectrometry. Both cell lines produced high-mannose N-glycans, mainly Man5 (80 and 86%, respectively) and Man4 (16 and 11%, respectively). The high degree of N-glycan homogeneity and the high-mannose N-glycosylation profile of these BY-2 cell lines is an asset for their use as expression platforms.

scholarly journals Alamethicin permeabilizes the plasma membrane and mitochondria but not the tonoplast in tobacco (Nicotiana tabacum L. cv Bright Yellow) suspension cells

2005 ◽  
Vol 389 (3) ◽  
pp. 695-704 ◽  
Author(s):  
Sandra Matic ◽  
Daniela A. Geisler ◽  
Ian M. Møller ◽  
Susanne Widell ◽  
Allan G. Rasmusson
Keyword(s):  
Nicotiana Tabacum ◽  
Plasma Membranes ◽  
Animal Cells ◽  
Suspension Cells ◽  
Respiratory Enzymes ◽  
Mitochondrial Oxygen Consumption ◽  
Permeabilized Cells ◽  
Bright Yellow ◽  
Transport Chain ◽  
Nicotiana Tabacum L

The ion channel-forming peptide AlaM (alamethicin) is known to permeabilize isolated mitochondria as well as animal cells. When intact tobacco (Nicotiana tabacum L.) Bright Yellow-2 cells were treated with AlaM, the cells became permeable for low-molecular-mass molecules as shown by induced leakage of NAD(P)+. After the addition of cofactors and substrates, activities of cytosolic as well as mitochondrial respiratory enzymes could be directly determined inside the permeabilized cells. However, at an AlaM concentration at which the cytoplasmic enzymes were maximally accessible, the vacuole remained intact, as indicated by an unaffected tonoplast proton gradient. Low-flux permeabilization of plasma membranes and mitochondria at moderate AlaM concentrations was reversible and did not affect cell vigour. Higher AlaM concentrations induced cell death. After the addition of catalase that removes the H2O2 necessary for NADH oxidation by apoplastic peroxidases, mitochondrial oxygen consumption could be measured in permeabilized cells. Inhibitor-sensitive oxidation of the respiratory substrates succinate, malate and NADH was observed after the addition of the appropriate coenzymes (ATP, NAD+). The capacities of different pathways in the respiratory electron-transport chain could thus be determined directly. We conclude that AlaM permeabilization provides a very useful tool for monitoring metabolic pathways or individual enzymes in their native proteinaceous environment with controlled cofactor concentrations. Possible uses and limitations of this method for plant cell research are discussed.

scholarly journals The Anti-Tumor Activity of the NEDD8 Inhibitor Pevonedistat in Neuroblastoma

2021 ◽  
Vol 22 (12) ◽  
pp. 6565
Author(s):  
Jennifer H. Foster ◽  
Eveline Barbieri ◽  
Linna Zhang ◽  
Kathleen A. Scorsone ◽  
Myrthala Moreno-Smith ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Wild Type ◽  
Tumor Weight ◽  
Cycle Arrest ◽  
P53 Status ◽  
Neuroblastoma Cell Lines ◽  
Anti Tumor Activity

Pevonedistat is a neddylation inhibitor that blocks proteasomal degradation of cullin–RING ligase (CRL) proteins involved in the degradation of short-lived regulatory proteins, including those involved with cell-cycle regulation. We determined the sensitivity and mechanism of action of pevonedistat cytotoxicity in neuroblastoma. Pevonedistat cytotoxicity was assessed using cell viability assays and apoptosis. We examined mechanisms of action using flow cytometry, bromodeoxyuridine (BrDU) and immunoblots. Orthotopic mouse xenografts of human neuroblastoma were generated to assess in vivo anti-tumor activity. Neuroblastoma cell lines were very sensitive to pevonedistat (IC50 136–400 nM). The mechanism of pevonedistat cytotoxicity depended on p53 status. Neuroblastoma cells with mutant (p53MUT) or reduced levels of wild-type p53 (p53si-p53) underwent G2-M cell-cycle arrest with rereplication, whereas p53 wild-type (p53WT) cell lines underwent G0-G1 cell-cycle arrest and apoptosis. In orthotopic neuroblastoma models, pevonedistat decreased tumor weight independent of p53 status. Control mice had an average tumor weight of 1.6 mg + 0.8 mg versus 0.5 mg + 0.4 mg (p < 0.05) in mice treated with pevonedistat. The mechanism of action of pevonedistat in neuroblastoma cell lines in vitro appears p53 dependent. However, in vivo studies using mouse neuroblastoma orthotopic models showed a significant decrease in tumor weight following pevonedistat treatment independent of the p53 status. Novel chemotherapy agents, such as the NEDD8-activating enzyme (NAE) inhibitor pevonedistat, deserve further study in the treatment of neuroblastoma.

scholarly journals Phosphoglycolate has profound metabolic effects but most likely no role in a metabolic DNA response in cancer cell lines

2019 ◽  
Vol 476 (4) ◽  
pp. 629-643 ◽  
Author(s):  
Isabelle Gerin ◽  
Marina Bury ◽  
Francesca Baldin ◽  
Julie Graff ◽  
Emile Van Schaftingen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Oxidative Dna Damage ◽  
Fold Increase ◽  
Metabolic Effects ◽  
Phosphoglycerate Mutase ◽  
Wild Type ◽  
Metabolic Disturbances ◽  
Phosphoglycolate Phosphatase ◽  
Damage Repair

Abstract Repair of a certain type of oxidative DNA damage leads to the release of phosphoglycolate, which is an inhibitor of triose phosphate isomerase and is predicted to indirectly inhibit phosphoglycerate mutase activity. Thus, we hypothesized that phosphoglycolate might play a role in a metabolic DNA damage response. Here, we determined how phosphoglycolate is formed in cells, elucidated its effects on cellular metabolism and tested whether DNA damage repair might release sufficient phosphoglycolate to provoke metabolic effects. Phosphoglycolate concentrations were below 5 µM in wild-type U2OS and HCT116 cells and remained unchanged when we inactivated phosphoglycolate phosphatase (PGP), the enzyme that is believed to dephosphorylate phosphoglycolate. Treatment of PGP knockout cell lines with glycolate caused an up to 500-fold increase in phosphoglycolate concentrations, which resulted largely from a side activity of pyruvate kinase. This increase was much higher than in glycolate-treated wild-type cells and was accompanied by metabolite changes consistent with an inhibition of phosphoglycerate mutase, most likely due to the removal of the priming phosphorylation of this enzyme. Surprisingly, we found that phosphoglycolate also inhibits succinate dehydrogenase with a Ki value of <10 µM. Thus, phosphoglycolate can lead to profound metabolic disturbances. In contrast, phosphoglycolate concentrations were not significantly changed when we treated PGP knockout cells with Bleomycin or ionizing radiation, which are known to lead to the release of phosphoglycolate by causing DNA damage. Thus, phosphoglycolate concentrations due to DNA damage are too low to cause major metabolic changes in HCT116 and U2OS cells.

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

scholarly journals CYP1A1 and CYP1A2 expression: Comparing ‘humanized’ mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

2009 ◽  
Vol 237 (1) ◽  
pp. 119-126 ◽  
Author(s):  
Shigeyuki Uno ◽  
Kaori Endo ◽  
Yuji Ishida ◽  
Chise Tateno ◽  
Makoto Makishima ◽  
...  
Keyword(s):  
Cell Lines ◽  
Wild Type ◽  
Humanized Mouse ◽  
Mouse Hepatoma ◽  
Human And Mouse ◽  
Mouse Lines

scholarly journals DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1.

1994 ◽  
Vol 125 (3) ◽  
pp. 625-638 ◽  
Author(s):  
J Lukas ◽  
H Müller ◽  
J Bartkova ◽  
D Spitkovsky ◽  
A A Kjerulff ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Complex Formation ◽  
Cyclin D1 ◽  
Cell Lines ◽  
T Antigen ◽  
Regulatory Function ◽  
Retinoblastoma Gene ◽  
Checkpoint Control ◽  
Wild Type ◽  
In Cells

The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.

scholarly journals NtRNF217, Encoding a Putative RBR E3 Ligase Protein of Nicotiana tabacum, Plays an Important Role in the Regulation of Resistance to Ralstonia solanacearum Infection

2021 ◽  
Vol 22 (11) ◽  
pp. 5507
Author(s):  
Ying Liu ◽  
Yuanman Tang ◽  
Xi Tan ◽  
Wei Ding
Keyword(s):  
Nicotiana Tabacum ◽  
Ralstonia Solanacearum ◽  
Plant Immunity ◽  
E3 Ligase ◽  
Marker Genes ◽  
Susceptible Cultivar ◽  
Wild Type ◽  
Disease Symptoms ◽  
Short Time ◽  
Resistance To Ralstonia Solanacearum

E3 ubiquitin ligases, the most important part of the ubiquitination process, participate in various processes of plant immune response. RBR E3 ligase is one of the E3 family members, but its functions in plant immunity are still little known. NtRNF217 is a RBR E3 ligase in tobacco based on the sequence analysis. To assess roles of NtRNF217 in tobacco responding to Ralstonia solanacearum, overexpression experiments in Nicotiana tabacum (Yunyan 87, a susceptible cultivar) were performed. The results illuminated that NtRNF217-overexpressed tobacco significantly reduced multiplication of R. solanacearum and inhibited the development of disease symptoms compared with wild-type plants. The accumulation of H2O2 and O2− in NtRNF217-OE plants was significantly higher than that in WT-Yunyan87 plants after pathogen inoculation. The activities of CAT and SOD also increased rapidly in a short time after R. solanacearum inoculation in NtRNF217-OE plants. What is more, overexpression of NtRNF217 enhanced the transcript levels of defense-related marker genes, such as NtEFE26, NtACC Oxidase, NtHIN1, NtHSR201, and NtSOD1 in NtRNF217-OE plants after R. solanacearum inoculation. The results suggested that NtRNF217 played an important role in regulating the expression of defense-related genes and the antioxidant enzymes, which resulted in resistance to R. solanacearum infection.

scholarly journals Cytotoxic Efficacy and Resistance Mechanism of a TRAIL and VEGFA-Peptide Fusion Protein in Colorectal Cancer Models

2021 ◽  
Vol 22 (6) ◽  
pp. 3160
Author(s):  
Michal Kopczynski ◽  
Malgorzata Statkiewicz ◽  
Magdalena Cybulska ◽  
Urszula Kuklinska ◽  
Katarzyna Unrug-Bielawska ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Transmembrane Protein ◽  
Acquired Resistance ◽  
Protein A ◽  
Resistance Mechanism ◽  
Receptor Expression ◽  
Human Colorectal Cancer ◽  
Protein Activity ◽  
Anticancer Efficacy

TNF-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein capable of selectively inducing apoptosis in cancer cells by binding to its cognate receptors. Here, we examined the anticancer efficacy of a recently developed chimeric AD-O51.4 protein, a TRAIL fused to the VEGFA-originating peptide. We tested AD-O51.4 protein activity against human colorectal cancer (CRC) models and investigated the resistance mechanism in the non-responsive CRC models. The quantitative comparison of apoptotic activity between AD-O51.4 and the native TRAIL in nine human colorectal cancer cell lines revealed dose-dependent toxicity in seven of them; the immunofluorescence-captured receptor abundance correlated with the extent of apoptosis. AD-O51.4 reduced the growth of CRC patient-derived xenografts (PDXs) with good efficacy. Cell lines that acquired AD-O51.4 resistance showed a significant decrease in surface TRAIL receptor expression and apoptosis-related proteins, including Caspase-8, HSP60, and p53. These results demonstrate the effectiveness of AD-O51.4 protein in CRC preclinical models and identify the potential mechanism underlying acquired resistance. Progression of AD-O51.4 to clinical trials is expected.

Mutation at autosomal loci of Chinese hamster ovary cells: involvement of a high-frequency event silencing two linked alleles

1983 ◽  
Vol 3 (7) ◽  
pp. 1172-1181
Author(s):  
W E Bradley
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
High Frequency ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Low Frequency ◽  
Class Ii ◽  
Class I ◽  
Wild Type ◽  
Ovary Cells

Two classes of cell lines heterozygous at the galactokinase (glk) locus have been isolated from Chinese hamster ovary cells. Class I, selected by plating nonmutagenized wild-type cells at low density in medium containing 2-deoxygalactose at a partially selective concentration, underwent subsequent mutation to the glk-/- genotype at a low frequency (approximately 10(-6) per cell), which was increased by mutagenesis. Class II heterozygotes, isolated by sib selection from mutagenized wild-type cells, had a higher spontaneous frequency of mutation to the homozygous state (approximately 10(-4) per cell), which was not affected by mutagenesis. About half of the glk-/- mutants derived from a class II heterozygote, but not the heterozygote itself, were functionally hemizygous at the syntenic thymidine kinase (tk) locus. Similarly, a tk+/- heterozygote with characteristics analogous to the class II glk+/- cell lines underwent high-frequency mutation to tk-/-, and most of these mutants, but not the tk+/- heterozygote, were functionally hemizygous at the glk locus. A model is proposed, similar to that for the mutational events at the adenine phosphoribosyl transferase locus (W. E. C. Bradley and D. Letovanec, Somatic Cell Genet. 8:51-66, 1982), of two different events, high and low frequency, being responsible for mutation at either of the linked loci tk and glk. The low-frequency event may be a point mutation, but the high-frequency event, in many instances, involves coordinated inactivation of a portion of a chromosome carrying the two linked alleles. Class II heterozygotes would be generated as a result of a low-frequency event at one allele, and class I heterozygotes would be generated by a high-frequency event. Supporting this model was the demonstration that all class I glk+/- lines examined were functionally hemizygous at tk.

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