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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

2011; Elsevier BV; Volume: 723; Issue: 1 Linguagem: Inglês

10.1016/j.mrgentox.2011.03.016

1879-3592

Takafumi Kimoto, Kumiko Suzuki, Xiao mei Kobayashi, Vasily N. Dobrovolsky, Robert H. Heflich, Daishiro Miura, Yoshinori Kasahara,

Blood disorders and treatments

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 100 mg/kg N-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.