The in vitro PIG-A gene mutation assay: glycosylphosphatidylinositol (GPI)-related genotype-to-phenotype relationship in TK6 cells.
In our previous work, we established an in vitro variant of the currently developed in vivo PIG-A assay as promising mutagenicity test system. We applied the human B-lymphoblastoid cell line TK6 for the in vitro assay development, which is based on the cellular glycosylphosphatidylinositol (GPI) status. At least 22 genes are involved in GPI biosynthesis, leading to the complex situation that, in principle, multiple genes could induce a GPI-deficient phenotype by acquiring inactivating mutations. However, only the PIG-A gene is located on the X-chromosome, rendering PIG-A more sensitive compared to autosomal linked, GPI-relevant genes. In this work, we investigated the GPI-related genotype-to-phenotype relationship in TK6 cells. By a next-generation sequencing approach, we identified a heterozygous chromosomal deletion on chromosome 17, where the PIG-L gene is located. In the analyzed TK6 cell clones, the GPI-deficient phenotype was induced either by mutations in PIG-A, by the complete absence of PIG-A mRNA, or by deletions in the remaining functional PIG-L gene, causing loss of heterozygosity. The identified PIG-L heterozygosity could also be responsible for the increased sensitivity toward mutagenic ethyl methanesulfonate or UV-C treatments of p53-proficient TK6 compared to the TK6-related, but p53-deficient WI-L2-NS cell line. Moreover, the WI-L2-NS cell line was found to exhibit a much lower number of GPI-deficient mutant cells in the purchased cell batch, and WI-L2-NS exerted a lower spontaneous rate of GPI deficiency compared to TK6 cells.
Food Chemistry and Toxicology, Institute of Applied Biosciences (IAB), Karlsruhe Institute of Technology (KIT), Adenauerring 20a, 76131, Karlsruhe, Germany.
Christopher T
CT
Krüger
Food Chemistry and Toxicology, Institute of Applied Biosciences (IAB), Karlsruhe Institute of Technology (KIT), Adenauerring 20a, 76131, Karlsruhe, Germany.
Bettina M
BM
Fischer
Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Postfach 3640, 76021, Karlsruhe, Germany.
Olivier
O
Armant
Chair of Biological Chemistry, Technische Universität München, 85350, Freising-Weihenstephan, Germany.
Volker
V
Morath
Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Postfach 3640, 76021, Karlsruhe, Germany.
Uwe
U
Strähle
Food Chemistry and Toxicology, Institute of Applied Biosciences (IAB), Karlsruhe Institute of Technology (KIT), Adenauerring 20a, 76131, Karlsruhe, Germany. andrea.hartwig@kit.edu.
Andrea
A
Hartwig
D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002872:Chromosome Deletion; D002886:Chromosomes, Human, Pair 17 / Q000235:genetics; D004252:DNA Mutational Analysis; D004587:Electrophoresis, Agar Gel; D005020:Ethyl Methanesulfonate / Q000633:toxicity; D005434:Flow Cytometry; D056726:Genetic Association Studies; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis* / Q000172:deficiency / Q000235:genetics; D006579:Heterozygote; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009152:Mutagenicity Tests / Q000379:methods*; D009154:Mutation / Q000187:drug effects / Q000528:radiation effects; D014466:Ultraviolet Rays / Q000009:adverse effects
D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; C080086:phosphatidylinositol glycan-class A protein; D005020:Ethyl Methanesulfonate
GPI biosynthesis; In vitro mutation assay; Next-generation sequencing; PIG-A; PIG-L; TK6