scholarly journals Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing

2020 ◽  
Vol 175 (2) ◽  
pp. 251-265 ◽  
Author(s):  
Xilin Li ◽  
Si Chen ◽  
Xiaoqing Guo ◽  
Qiangen Wu ◽  
Ji-Eun Seo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Expression System ◽  
Cytochrome P450s ◽  
Mass Spectrometry Analysis ◽  
Established Cell Line ◽  
Micronucleus Formation ◽  
Tk6 Cells ◽  
Genotoxicity Testing

Abstract Metabolism plays a key role in chemical genotoxicity; however, most mammalian cells used for in vitro genotoxicity testing lack effective metabolizing enzymes. We recently developed a battery of TK6-derived cell lines that individually overexpress 1 of 8 cytochrome P450s (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, and 3A4) using a lentiviral expression system. The increased expression and metabolic function of each individual CYP in each established cell line were confirmed using real-time PCR, Western blotting, and mass spectrometry analysis; the parental TK6 cells and empty vector (EV) transduced cells had negligible CYP levels. Subsequently, we evaluated these cell lines using 2 prototypical polyaromatic hydrocarbon mutagens, 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P), that require metabolic activation to exert their genotoxicity. DMBA-induced cytotoxicity, phosphorylation of histone H2A.X, and micronucleus formation were significantly increased in TK6 cells with CYP1A1, 1B1, 2B6, and 2C19 expression as compared with EV controls. B[a]P significantly increased cytotoxicity, DNA damage, and chromosomal damage in TK6 cells overexpressing CYP1A1 and 1B1 when compared with EV controls. B[a]P also induced micronucleus formation in TK6 cells expressing CYP1A2. These results suggest that our CYP-expressing TK6 cell system can be used to detect the genotoxicity of compounds requiring metabolic transformation.

scholarly journals Poly-L-ornithine-mediated transformation of mammalian cells.

1987 ◽  
Vol 7 (6) ◽  
pp. 2286-2293 ◽  
Author(s):  
V C Bond ◽  
B Wold
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Protein Product ◽  
Marker Genes ◽  
Recipient Mouse ◽  
L Cells ◽  
Dna And Rna ◽  
Selectable Marker Genes

Poly-L-ornithine has been used to introduce DNA and RNA into mammalian cells in culture. Ornithine-mediated DNA transfer has several interesting and potentially useful properties. The procedure is technically straightforward and is easily applied to either small or large numbers of recipient cells. The efficiency of transformation is high. Under optimal conditions, 1 to 2% of recipient mouse L cells take up and continue to express selectable marker genes. DNA content of transformants can be varied reproducibly, yielding cells with just one or two copies of the new gene under one set of conditions, while under a different set of conditions 25 to 50 copies are acquired. Cotransformation and expression of physically unlinked genes occur at high efficiency under conditions favoring multiple-copy transfer. Polyornithine promotes gene transfer into cell lines other than L cells. These include Friend erythroleukemia cells and NIH 3T3 cells. Both are transformed about 1 order of magnitude more efficiently by this procedure than by standard calcium phosphate products. However, the method does not abolish the large transformation efficiency differences between these cell lines that have been observed previously by other techniques. (vi) mRNA synthesized in vitro was also introduced into cells by this method. The RNA was translated resulting in a transient accumulation of the protein product.

scholarly journals RPNs Levels are Prognostic and Diagnostic Markers for Hepatocellular Carcinoma

2021 ◽  
Author(s):  
Wangyang zheng ◽  
Yuling Zheng ◽  
Xue Bai ◽  
Yongxu Zhou ◽  
Liang Yu ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Tumor Grade ◽  
Ubiquitin Proteasome System ◽  
Functional Enrichment ◽  
Migration And Invasion ◽  
Regulatory Subunits ◽  
Hcc Cells

Abstract Background: Ribophorin family (RPNs) are important regulatory subunits of the proteasome. By influencing Ubiquitin-proteasome system activity, RPNs are responsible for almost all processes of physiology and pathology of mammalian cells. Nevertheless, little is known about the role of RPNs in HCC.Methods: In this work, using the online databases Oncomine, UCSC, Kaplan-Meier Plotter, UALCAN, cBioPortal, TIMER2, GeneMANIA,and STRING, we first evaluated the expression, diagnostic, prognostic, genetic alteration, immunity, gene network, and functional enrichment of RPNs in HCC. QPCR and western blot were used to detect RPN6 and RPN9 expressions in HCC tissues and cell lines. Then we performed studies to eveulated their functions in HCC cells proliferation, migration, and invasion in vitro. Results: All RPNs were surprisingly consistently upregulated in HCC tissues. Moreover, RPNs expression pattern is correlated with HCC tumor grade. RPN2, RPN3, RPN6, RPN9, RPN10, RPN11, and RPN12 have robust values in HCC diagnose. Then, survival analysis revealed that high expression of RPN1, RPN2, RPN4, RPN5, RPN6, RPN9, and RPN11were correlated with unfavorable HCC overall survival. Functional enrichment for RPNs, indicated that RPNs have many potential biosynthesis activities expert for UPS functions. Western blot, and qRT-PCR further verified these results in HCC tissues and cell lines. The silencing of RPN6 and RPN9 significantly influenced HCC cells' proliferation, migration, and invasion in vitro.Conclusions: RPN families functions as an important oncogene in HCC. RPN6 and RPN9 have the potential to be potential biomarkers and targets for HCC.

Attachment of Salmonella to mammalian cells in vitro

1983 ◽  
Vol 29 (12) ◽  
pp. 1731-1735 ◽  
Author(s):  
Clifford S. Mintz ◽  
Dean O. Cliver ◽  
R. H. Deibel
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Culture Cell ◽  
Intestinal Cell ◽  
Tissue Culture Cell ◽  
Bacterial Cells ◽  
Intestinal Cell Line ◽  
Hela Cell Lines ◽  
High Concentrations

The attachment of Salmonella typhimurium strain PHL67342 to several mammalian tissue culture cell lines was investigated. Strain PHL67342 failed to attach in significant numbers to the Buffalo green monkey (BGM), swine testicular (ST), and HeLa cell lines. Significant attachment was observed with the Henle intestinal cell line. Log-phase cells of strain PHL67342 attached in greatest numbers to the Henle cells after 45 min of incubation at 37 °C. Attachment to the Henle cells was not affected by D-mannose or D-galactose, but was markedly inhibited by high concentrations of alpha-methyl-D-mannoside. Also, Salmonella lipopolysaccharide had no effect on the attachment of strain PHL67342 to the Henle cells. Fimbriae were not detected on the bacterial cells used in the adherence experiments. These results suggest that some bacterial factor(s) other than fimbriae and lipopolysaccharide mediate the attachment of strain PHL67342 to the Henle cells.

scholarly journals CK2-mediated phosphorylation of Che-1/AATF is required for its pro-proliferative activity

2021 ◽  
Author(s):  
Valeria Catena ◽  
Tiziana Bruno ◽  
Simona Iezzi ◽  
Silvia Matteoni ◽  
Annalisa Salis ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Histone Acetylation ◽  
Rna Polymerase Ii ◽  
Cell Lines ◽  
Human Cancer ◽  
Histone H3 ◽  
Histone Deacetylation ◽  
Site Directed Mutagenesis ◽  
Mass Spectrometry Analysis

Abstract Background Che-1/AATF (Che-1) is an RNA polymerase II binding protein involved in several cellular processes, including proliferation, apoptosis and response to stress. We have recently demonstrated that Che-1 is able to promote cell proliferation by sustaining global histone acetylation in Multiple Myeloma (MM) cells where it interacts with histone proteins and competes with HDAC class I members for binding. Methods Site-directed Mutagenesis was performed to generate a Che-1 mutant (Che-1 3S) lacking three-serine residue (Ser316, Ser320 and Ser321) in 308–325 AA region. Western blot experiments were conducted to examine the effect of depletion or over-expression of Che-1 and Che-1 3S mutant on histone acetylation, in different human cancer cell lines. Proliferation assays were assessed to estimate the change in cells number when Che-1 was over-expressed or deleted. Immunoprecipitation assays were performed to evaluate Che-1/histone H3 interaction when Ser316, Ser320 and Ser321 were removed. The involvement of CK2 kinase in Che-1 phosphorylation at these residues was analysed by in vitro kinase, 2D gel electrophoresis assays and mass spectrometry analysis. Results Here, we confirm that Che-1 depletion reduces cell proliferation with a concomitant general histone deacetylation in several tumor cell lines. Furthermore, we provided evidence that CK2 protein kinase phosphorylates Che-1 at Ser316, Ser320 and Ser321 and that these modifications are required for Che-1/histone H3 binding. These results improve our understanding onto the mechanisms by which Che-1 regulates histone acetylation and cell proliferation. Conclusions Che-1 phosphorylation at Ser316, Ser320 and Ser321 by CK2 promotes the interaction with histone H3 and represents an essential requirement for Che-1 pro-proliferative ability.

scholarly journals SUV39H1-DNMT3A-mediated epigenetic regulation of Tim-3 and galectin-9 in the cervical cancer

2020 ◽  
Author(s):  
Li Zhang ◽  
Sijuan Tian ◽  
Minyi Zhao ◽  
Ting Yang ◽  
Shimin Quan ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cell Lines ◽  
Epigenetic Regulation ◽  
Promoter Region ◽  
Expression System ◽  
Methylation Status ◽  
Regulation Mechanism ◽  
Cellular Expression

Abstract Background: Methylation of histone 3 at lysine 9 (H3K9) and DNA methylation are epigenetic marks correlated with genes silencing. The tumor microenvironment significantly influences therapeutic responses and clinical outcomes. The epigenetic-regulation mechanism of the costimulatory factors Tim-3 and galectin-9 in cervical cancer remains unknown. Methods: The methylation status of HAVCR2 and LGALS9 were detected by MS-PCR in cervical cancer tissues and cell lines. The underlying molecular mechanism of SUV39H1-DNMT3A-Tim-3/galectin-9 regulation was elucidated using cervical cancer cell lines containing siRNA or/and over-expression system. Confirmation of the regulation of DNMT3A by SUV39H1 used ChIP-qPCR.Results: SUV39H1 up-regulates H3K9me3 expression at the DNMT3A promoter region, which in turn induced expression of DNMT3A in cervical cancer. In addition, the mechanistic studies indicate that DNMT3A mediates the epigenetic modulation of the HAVCR2 and LGALS9 genes by directly binding to their promoter regions in vitro. Moreover, in an in vivo assay, the expression profile of SUV39H1 up-regulates the level of H3K9me3 at the DNMT3A promoter region was found to correlate with Tim-3 and galectin-9 cellular expression level. Conclusion: These results indicate that SUV39H1-DNMT3A is a crucial Tim-3 and galectin-9 regulatory axis in cervical cancer.

scholarly journals Cytotoxic Phenanthrene, Dihydrophenanthrene, and Dihydrostilbene Derivatives and Other Aromatic Compounds from Combretum laxum

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3154
Author(s):  
Eder Bisoli ◽  
Talita Vilalva Freire ◽  
Nídia Cristiane Yoshida ◽  
Walmir Silva Garcez ◽  
Lyara Meira Marinho Queiróz ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Mammalian Cells ◽  
Human Cancer ◽  
Radical Scavenging ◽  
Cancer Cell Lines ◽  
In Vitro Cytotoxicity ◽  
Mass Spectrometry Data ◽  
Spectroscopic Techniques

The chemical investigation of the roots and stems of Combretum laxum yielded a new dihydrostilbene derivative, 4′-hydroxy-3,3′,4-trimethoxy-5-(3,4,5-trimethoxyphenoxy)-bibenzyl (1), two phenanthrenes (2–3), and three dihydrophenanthrenes (4–6), along with one lignan, three triterpenoids, one aurone, one flavone, one naphthoquinone, and two benzoic acid derivatives. Their structures were determined by 1D and 2D nuclear magnetic resonance (NMR) spectroscopic techniques and/or mass spectrometry data. The occurrence of dihydrostilbenoid, phenanthrene and dihydrophenanthrene derivatives is unprecedented in a Combretum species native to the American continent. 2,7-Dihydroxy-4,6-dimethoxyphenanthrene, 2,6-dihydroxy-4,7-dimethoxy-9,10-dihydrophenanthrene and 5-O-methyl apigenin are novel findings in the Combretaceae, as is the isolation of compounds belonging to the chemical classes of aurones and naphthoquinones, while (+)-syringaresinol is reported for the first time in the genus Combretum. Compounds 1–6 were also evaluated for their in vitro cytotoxicity against five human cancer cell lines, and radical-scavenging ability against 1,1-diphenyl-2-picryl-hydrazyl (DPPH). 6-Methoxycoelonin (4) was the most cytotoxic against melanoma cells (IC50 2.59 ± 0.11 µM), with a high selectivity index compared with its toxicity against nontumor mammalian cells (SI 25.1). Callosin (6), despite exhibiting the strongest DPPH-scavenging activity (IC50 17.7 ± 0.3 µM), proved marginally inhibitory to the five cancer cell lines tested, indicating that, at least for these cells, antioxidant potential is unrelated to antiproliferative activity.

Construction of novel antibodies by use of DNA transfection: design of plasmid vectors

1986 ◽  
Vol 317 (1540) ◽  
pp. 425-432 ◽  
Keyword(s):  
Cell Lines ◽  
Dna Sequences ◽  
Lymphoid Cell ◽  
Expression System ◽  
Immunoglobulin Gene ◽  
Dna Transfection ◽  
Efficient Expression ◽  
Lymphoid Cell Lines ◽  
Antibody Gene

The DNA that encodes antibodies can be manipulated in vitro and reintroduced into lymphoid cell lines. In this way, lymphocyte transfectants can be established which secrete milligram quantities of novel antibody molecules. Here we present data concerning the DNA sequences that are needed for efficient expression of the transfected antibody gene and give examples of the way in which this expression system for immunoglobulin gene DNA can be used for the production of useful antibody-related molecules.

scholarly journals Nuclear Localization of Japanese Encephalitis Virus Core Protein Enhances Viral Replication

2005 ◽  
Vol 79 (6) ◽  
pp. 3448-3458 ◽  
Author(s):  
Yoshio Mori ◽  
Tamaki Okabayashi ◽  
Tetsuo Yamashita ◽  
Zijiang Zhao ◽  
Takaji Wakita ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Cell Lines ◽  
Japanese Encephalitis ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Core Protein ◽  
Encephalitis Virus ◽  
Wild Type ◽  
The Core

ABSTRACT Japanese encephalitis virus (JEV) core protein was detected in both the nucleoli and cytoplasm of mammalian and insect cell lines infected with JEV or transfected with the expression plasmid of the core protein. Mutation analysis revealed that Gly42 and Pro43 in the core protein are essential for the nuclear and nucleolar localization. A mutant M4243 virus in which both Gly42 and Pro43 were replaced by Ala was recovered by plasmid-based reverse genetics. In C6/36 mosquito cells, the M4243 virus exhibited RNA replication and protein synthesis comparable to wild-type JEV, whereas propagation in Vero cells was impaired. The mutant core protein was detected in the cytoplasm but not in the nucleus of either C6/36 or Vero cell lines infected with the M4243 virus. The impaired propagation of M4243 in mammalian cells was recovered by the expression of wild-type core protein in trans but not by that of the mutant core protein. Although M4243 mutant virus exhibited a high level of neurovirulence comparable to wild-type JEV in spite of the approximately 100-fold-lower viral propagation after intracerebral inoculation to 3-week-old mice of strain Jcl:ICR, no virus was recovered from the brain after intraperitoneal inoculation of the mutant. These results indicate that nuclear localization of JEV core protein plays crucial roles not only in the replication in mammalian cells in vitro but also in the pathogenesis of encephalitis induced by JEV in vivo.

In vitro expression of a mouse tissue specific glutathione-peroxidase-like protein lacking the selenocysteine can protect stably transfected mammalian cells against oxidative damage

1996 ◽  
Vol 74 (1) ◽  
pp. 125-131 ◽  
Author(s):  
Patrick Vernet ◽  
Nicole Rigaudiére ◽  
Norbert Ghyselinck ◽  
Jean Pierre Dufaure ◽  
Joël R. Drevet
Keyword(s):  
Glutathione Peroxidase ◽  
Mammalian Cells ◽  
Expression System ◽  
Complete Sequence ◽  
Protein Domain ◽  
Mouse Tissue ◽  
Reading Frame ◽  
Mammalian Expression ◽  
Mouse Epididymis

The complete sequence of the mouse epididymal protein (MEP24) was cloned. It contains a 663 bp open-reading frame that, after conceptual translation, shows extensive identity with proteins belonging to the glutathione peroxidase (GPX) family. However, a major difference between GPX5 (MEP24) and other known GPXs concerns a protein domain known to be critical for GPX function. To find out what could be the physiological function of such a protein in the mouse epididymis, we have used a mammalian expression system to overexpress the GPX5 protein. Cells constitutively expressing the GPX5 protein were generated and assayed for their ability to metabolize regular substrates of GPX enzymes. Data presented here show that the GPX5-expressing cells can metabolize hydrogen peroxide in a manner that is consistent with a peroxidase activity. However, the substrate preference of the GPX5-expressing cells and their apparent insensitivity to a regular inhibitor of GPX enzymes suggest that the GPX5 protein belongs to a particular class of GPX proteins. Involvement of this protein in the physiology of the mouse epididymis is discussed.Key words: glutathione peroxidase (GPX), mouse epididymis, MEP24, GPX5 cDNA, selenocysteine, GPX5 polyclonal antibody, spermatozoa, CHO-KI cells.

In-vitro and in-vivo consequences of mutations in the von Willebrand factor cleaving protease ADAMTS13 in thrombotic thrombocytopenic purpura

2006 ◽  
Vol 96 (10) ◽  
pp. 454-464 ◽  
Author(s):  
Roberta Donadelli ◽  
Federica Banterla ◽  
Miriam Galbusera ◽  
Cristina Capoferri ◽  
Sara Bucchioni ◽  
...  
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Von Willebrand Factor ◽  
Protease Activity ◽  
Mammalian Cells ◽  
Expression System ◽  
Thrombocytopenic Purpura ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThrombotic thrombocytopenic purpura (TTP) is a disease characterized by microvascular thrombosis, often associated with deficiency of the von Willebrand factor (VWF) cleaving protease ADAMTS13.We investigated the spectrum of ADAMTS13 gene mutations in patients with TTP and congenital ADAMTS13 deficiency to establish the consequences on ADAMTS13 processing and activity. We describe five missense (V88M, G1239V, R1060W, R1123C and R1219W), 1 nonsense (W1016Stop) and 1 insertion (82_83insT) mutations. In two patients no mutation was identified despite undetectable protease activity. Expression in HEK293 mammalian cells (V88M, G1239V, R1123C and R1219W) documented that three missense mutants were not secreted, whereas theV88M was secreted at low levels and with reduced activity. We also provide evidence that impaired secretion of ADAMTS13 mutants observed in vitro translates into severely reduced ADAMTS13 antigen levels in patients in vivo. To evaluate whether the small amounts of mutant protease present in the circulation of patients had VWF cleaving activity, WT and mutant rADAMTS13 were stably expressed in Drosophila S2 cells under the influence of the Drosophila BiP protein signal sequence, which allows protein secretion. Drosophila expression system showed a 40–60% protease activity in the mutants. Several single nucleotide polymorphisms (SNPs) within exons and intron boundaries were found in patients, suggesting that the interplay of SNPs could at least in part account for ADAMTS13 functional abnormalities in patients without mutations. In conclusion, defective secretion and impaired activity of the mutants concur to determine an almost complete deficiency of ADAMTS13 activity in patients with a homozygous or two heterozygous ADAMTS13 mutations.

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