CD59-deficient bone marrow erythroid cells from rats treated with procarbazine and propyl-nitrosourea have mutations in the Pig-a gene |
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Authors: | Javier R. Revollo Azra Dad Mason G. Pearce Roberta A. Mittelstaedt Andrea Casildo Rena G. Lapidus Timothy W. Robison Vasily N. Dobrovolsky |
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Affiliation: | 1. Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, Arkansas, USA;2. Greenbaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, USA;3. Division of Pulmonary, Allergy and Critical Care Products, Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, Maryland, USA |
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Abstract: | Procarbazine (PCZ) and N-propyl-N-nitrosourea (PNU) are rodent mutagens and carcinogens. Both induce GPI-anchored marker-deficient mutant-phenotype red blood cells (RBCs) in the flow cytometry-based rat RBC Pig-a assay. In the present study, we traced the origin of the RBC mutant phenotype by analyzing Pig-a mutations in the precursors of RBCs, bone marrow erythroid cells (BMEs). Rats were exposed to a total of 450 mg/kg PCZ hydrochloride or 300 mg/kg PNU, and bone marrow was collected 2, 7, and 10 weeks later. Using a flow cell sorter, we isolated CD59-deficient mutant-phenotype BMEs from PCZ- and PNU-treated rats and examined their endogenous X-linked Pig-a gene by next generation sequencing. Pig-a mutations consistent with the properties of PCZ and PNU were found in sorted mutant-phenotype BMEs. PCZ induced mainly A > T transversions with the mutated A on the nontranscribed strand of the Pig-a gene, while PNU induced mainly T > A transversions with the mutated T on the nontranscribed strand. The treatment-induced mutations were distributed across the protein coding sequence of the Pig-a gene. The causal relationship between BMEs and RBCs and the agent-specific mutational spectra in CD59-deicient BMEs indicate that the rat RBC Pig-a assay, scoring CD59-deficient mutant-phenotype RBCs in peripheral blood, detects Pig-a gene mutation. |
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Keywords: | flow cytometry glycosyl phosphatidylinositol red blood cells sequencing |
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