title: Development of an in vitro PIG-A gene mutation assay in human cells.
abstract: Mutagens can be carcinogens, and traditionally, they have been identified in vitro using the Salmonella 'Ames' reverse mutation assay. However, prokaryotic DNA packaging, replication and repair systems are mechanistically very different to those in the humans we inevitably seek to protect. Therefore, for many years, mammalian cell line genotoxicity assays that can detect eukaryotic mutagens as well as clastogens and aneugens have been used. The apparent lack of specificity in these largely rodent systems, due partly to their mutant p53 status, has contributed to the use of animal studies to resolve data conflicts. Recently, silencing mutations at the PIG-A locus have been demonstrated to prevent glycophosphatidylinositol (GPI) anchor synthesis and consequentially result in loss of GPI-anchored proteins from the cell's extracellular surface. The successful exploitation of this mutant phenotype in animal studies has triggered interest in the development of an analogous in vitro PIG-A mutation screening assay. This article describes the development of a robust assay design using metabolically active human cells. The assay includes viability and cell membrane integrity assessment and conforms to the future ideas of the 21st-century toxicology testing.
authors.affiliation: Institute of Life Science, Swansea University Medical School, Singleton Park, Swansea SA2 8PP, UK.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Rees
authors.affiliation: Gentronix Ltd BioHub at Alderley Park, Alderley Edge, Cheshire, SK10 4TF, UK.
authors.forename: Matthew
authors.initials: M
authors.lastname: Tate
authors.affiliation: GlaxoSmithKline R&D, Ware, Hertfordshire SG12 0DP, UK and.
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Lynch
authors.affiliation: Institute of Life Science, Swansea University Medical School, Singleton Park, Swansea SA2 8PP, UK.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Thornton
authors.affiliation: Institute of Life Science, Swansea University Medical School, Singleton Park, Swansea SA2 8PP, UK.
authors.forename: Gareth J
authors.initials: GJ
authors.lastname: Jenkins
authors.affiliation: Gentronix Ltd BioHub at Alderley Park, Alderley Edge, Cheshire, SK10 4TF, UK.|Faculty of Life Sciences, University of Manchester, Manchester M13 9PL, UK.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Walmsley
authors.affiliation: Institute of Life Science, Swansea University Medical School, Singleton Park, Swansea SA2 8PP, UK.
authors.forename: George E
authors.initials: GE
authors.lastname: Johnson
mesh_terms: D002460:Cell Line; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009152:Mutagenicity Tests / Q000379:methods*; D009154:Mutation
chemical_list: D008565:Membrane Proteins; C080086:phosphatidylinositol glycan-class A protein