An untargeted metabolomics strategy to measure differences in metabolite uptake and excretion by mammalian cell lines

AbstractIntroductionIt is widely but erroneously believed that drugs get into cells by passing through the phospholipid bilayer portion of the plasma and other membranes. Much evidence shows, however, that this is not the case, and that drugs cross biomembranes by hitchhiking on transporters for other natural molecules to which these drugs are structurally similar. Untargeted metabolomics can provide a method for determining the differential uptake of such metabolites.ObjectivesBlood serum contains many thousands of molecules and provides a convenient source of biologically relevant metabolites. Our objective was to measure them.MethodsWe develop an untargeted LC-MS/MS method to detect a broad range of compounds present in human serum. We apply this to the analysis of the time course of the uptake and secretion of metabolites in serum by several human cell lines, by analysing changes in the serum that represents the extracellular phase (the ‘exometabolome’ or metabolic footprint).ResultsOur method measures some 4,000-5,000 metabolic features in both ES+ and ES− modes. We show that the metabolic footprints of different cell lines differ greatly from each other.ConclusionOur new, 15-minute untargeted metabolome method allows for the robust and convenient measurement of differences in the uptake of serum compounds by cell lines following incubation in serum, and its relation to differences in transporter expression.

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An untargeted metabolomics strategy to measure differences in metabolite uptake and excretion by mammalian cell lines

Metabolomics ◽

10.1007/s11306-020-01725-8 ◽

2020 ◽

Vol 16 (10) ◽

Author(s):

Marina Wright Muelas ◽

Ivayla Roberts ◽

Farah Mughal ◽

Steve O’Hagan ◽

Philip J. Day ◽

...

Keyword(s):

Cell Lines ◽

Time Course ◽

Phospholipid Bilayer ◽

Untargeted Metabolomics ◽

Future Research ◽

Human Cell Lines ◽

Biologically Relevant ◽

Mammalian Cell Lines ◽

Multiple Cell ◽

Transporter Expression

Abstract Introduction It is widely but erroneously believed that drugs get into cells by passing through the phospholipid bilayer portion of the plasma and other membranes. Much evidence shows, however, that this is not the case, and that drugs cross biomembranes by hitchhiking on transporters for other natural molecules to which these drugs are structurally similar. Untargeted metabolomics can provide a method for determining the differential uptake of such metabolites. Objectives Blood serum contains many thousands of molecules and provides a convenient source of biologically relevant metabolites. Our objective was to detect and identify metabolites present in serum, but to also establish a method capable of measure their uptake and secretion by different cell lines. Methods We develop an untargeted LC-MS/MS method to detect a broad range of compounds present in human serum. We apply this to the analysis of the time course of the uptake and secretion of metabolites in serum by several human cell lines, by analysing changes in the serum that represents the extracellular phase (the ‘exometabolome’ or metabolic footprint). Results Our method measures some 4000–5000 metabolic features in both positive and negative electrospray ionisation modes. We show that the metabolic footprints of different cell lines differ greatly from each other. Conclusion Our new, 15-min untargeted metabolome method allows for the robust and convenient measurement of differences in the uptake of serum compounds by cell lines following incubation in serum. This will enable future research to study these differences in multiple cell lines that will relate this to transporter expression, thereby advancing our knowledge of transporter substrates, both natural and xenobiotic compounds.

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Cytoplasmic transfer of a determinant for chloramphenicol resistance between mammalian cell lines

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1978.0033 ◽

1978 ◽

Vol 201 (1142) ◽

pp. 73-85 ◽

Cited By ~ 8

Keyword(s):

Cell Lines ◽

Human Cell ◽

Mitochondrial Protein ◽

Human Cell Lines ◽

Resistant Variant ◽

Chloramphenicol Resistance ◽

Resistance Phenotype ◽

Whole Cells ◽

Mammalian Cell Lines ◽

Cell Line Hela

A genetic analysis of the resistance phenotype of a recently described chloramphenicol-resistant variant derived from the human cell line, HeLa (MC63), has been undertaken. Whole cells or enucleated fragments, produced by treatment with cytochalasin B, were fused with chloramphenicol-sensitive mouse, or human cells. Enucleated cells (cytoplasts) act as very efficient donors of the resistance phenotype in fusions with other human cell lines derived from HeLa. We conclude that chloramphenicol resistance is determined cytoplasmically. Transfer of resistance to unrelated human cell lines occurred at much lower frequency and we were unable to demonstrate transfer to mouse cells. An examination of mitochondrial protein synthesis in the fusion products of cytoplasts and whole cells suggested that mixed populations of mitochondria from both parental cells were maintained under the conditions of selection.

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Evaluation and Design of Genome-wide CRISPR/Cas9 Knockout Screens

10.1101/117341 ◽

2017 ◽

Author(s):

Traver Hart ◽

Amy Tong ◽

Katie Chan ◽

Jolanda Van Leeuwen ◽

Ashwin Seetharaman ◽

...

Keyword(s):

Cell Lines ◽

Human Cell ◽

Viral Vectors ◽

Human Cancer ◽

Gene Knockout ◽

Essential Genes ◽

Human Cell Lines ◽

Protein Coding ◽

Mammalian Cell Lines ◽

Genome Scale

AbstractThe adaptation of CRISPR/Cas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs), targeting human protein-coding genes and encoded in viral vectors, have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we re-analyze 17 genome-scale knockout screens in human cell lines from three research groups using three different genome-scale gRNA libraries, using the Bayesian Analysis of Gene Essentiality (BAGEL) algorithm to identify essential genes, to refine and expand our previously defined set of human core essential genes, from 360 to 684 genes. We use this expanded set of reference Core Essential Genes (CEG2), plus empirical data from six CRISPR knockout screens, to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.

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A gene signature for gold nanoparticle-exposed human cell lines

Toxicology Research ◽

10.1039/c4tx00181h ◽

2015 ◽

Vol 4 (2) ◽

pp. 365-375 ◽

Cited By ~ 4

Author(s):

Ruei-Yue Liang ◽

Hsin-Fang Tu ◽

Xiaotong Tan ◽

Yu-Shan Yeh ◽

Pin Ju Chueh ◽

...

Keyword(s):

Gold Nanoparticle ◽

Cell Lines ◽

Mammalian Cell ◽

Cdna Microarray ◽

Human Cell ◽

Gene Signature ◽

Human Cell Lines ◽

Biological Assays ◽

Mammalian Cell Lines

A unique four-gene signature for AuNP exposure was identified using the cDNA microarray and evaluated by qPCR and biological assays in mammalian cell lines.

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Size-Specific Copper Nanoparticle Cytotoxicity Varies between Human Cell Lines

International Journal of Molecular Sciences ◽

10.3390/ijms22041548 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1548

Author(s):

Ina Na ◽

David C. Kennedy

Keyword(s):

Cell Culture ◽

Cell Lines ◽

Copper Nanoparticles ◽

Human Cell ◽

Time Course ◽

Culture Media ◽

Critical Role ◽

Human Cell Lines ◽

Cell Culture Media ◽

Particle Stability

Commercially available copper nanoparticles of three different sizes were tested for cytotoxicity against three human cell lines using four different cytotoxicity assays. This array of data was designed to elucidate trends in particle stability, uptake, and cytotoxicity. The copper nanoparticles are not stable in cell culture media, and rapid changes over the time course of the assays play a critical role in the measured endpoints. Typically, the 40–60 nm particles tested were more cytotoxic than either smaller or larger particles. These particles were also taken up more readily by cells and exhibited different stability dynamics in cell culture media. This provides a good correlation between total cellular uptake of copper and cytotoxicity that may be directly linked to particle stability, though it is unclear why the intermediate-sized particles exhibited these unique properties when compared with both larger and smaller particles.

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