title: Manifestation of Pig-a mutant bone marrow erythroids and peripheral blood erythrocytes in mice treated with N-ethyl-N-nitrosourea: direct sequencing of Pig-a cDNA from bone marrow cells negative for GPI-anchored protein expression.
abstract: Our previous rat studies indicate that the endogenous Pig-a gene is a promising reporter of in vivo mutation and potentially useful as the basis for an in vivo genotoxicity assay. The function of the Pig-a protein in the synthesis of glycosylphosphatidyl inositol (GPI) anchors is conserved in variety of eukaryotic cells, including human and rodent cells, which implies that Pig-a mutants can be measured in a similar manner in different mammalian species. In the present study, we developed a flow cytometric Pig-a assay for rapidly measuring gene mutation in the mouse. An antibody to TER-119, a specific cell-surface marker of murine erythroid lineage, was used to identify erythrocytes in peripheral blood (PB) and erythroids in bone marrow (BM). An antibody to CD24, a GPI-anchored protein, was used to identify Pig-a mutants as CD24-negative cells. CD-1 mice were administered a single dose of 100mg/kgN-ethyl-N-nitrosourea (ENU), and PB and BM were collected at 1, 2, and 4 weeks after dosing. While the Pig-a mutant frequency (MF) in PB was increased moderately at 2 and 4 weeks after ENU dosing, the Pig-a MF in BM was strongly increased starting at 1 week after the dosing, with the elevated MF persisting for at least 4 weeks after the dosing. We also used flow cytometric sorting to isolate CD24-negative erythroids from the BM of ENU-treated mice. cDNA sequencing indicated that these cells have mutations in the Pig-a gene, with base-pair substitutions typical of ENU-induced mutation spectra. The results indicate that the Pig-a mutation assay can be adapted for measuring mutation in BM erythroids and PB of mice. Taken together, the data suggest that Pig-a mutants are fixed in the BM, where they further proliferate and differentiate; erythrocytes derived from these BM Pig-a mutants transit from the BM and accumulate in PB.
authors.affiliation: TEIJIN Pharma Limited, Hino, Tokyo, Japan. t.kimoto@teijin.co.jp
authors.forename: Takafumi
authors.initials: T
authors.lastname: Kimoto
authors.forename: Kumiko
authors.initials: K
authors.lastname: Suzuki
authors.forename: Xiao Mei
authors.initials: XM
authors.lastname: Kobayashi
authors.forename: Vasily N
authors.initials: VN
authors.lastname: Dobrovolsky
authors.forename: Robert H
authors.initials: RH
authors.lastname: Heflich
authors.forename: Daishiro
authors.initials: D
authors.lastname: Miura
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kasahara
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects*; D004912:Erythrocytes / Q000187:drug effects*; D005038:Ethylnitrosourea / Q000633:toxicity*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D009152:Mutagenicity Tests / Q000379:methods*; D013997:Time Factors
chemical_list: D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; C080086:phosphatidylinositol glycan-class A protein; D005038:Ethylnitrosourea