Wang Yiying, Revollo Javier, McKinzie Page, Pearce Mason G, Dad Azra, Yucesoy Berran, Rosenfeldt Hans, Heflich Robert H, Dobrovolsky Vasily N
Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas.
Division of Nonclinical Science, Center for Tobacco Products, U.S. Food and Drug Administration, Silver Spring, Maryland.
Environ Mol Mutagen. 2018 Jan;59(1):4-17. doi: 10.1002/em.22152. Epub 2017 Nov 2.
The X-linked Pig-a gene encodes an enzyme required for the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. Pig-a mutant cells fail to synthesize GPI and to express GPI-anchored protein markers (e.g., CD90) on their surface. Marker deficiency serves as a phenotypic indicator of Pig-a mutation in various in vivo assays. Here, we describe an in vitro Pig-a mutation assay in L5178YTk mouse lymphoma cells, in which mutant-phenotype cells are measured by flow cytometry using a fluorescent anti-CD90 antibody. Increased frequencies of CD90-deficient mutants were detected in cells treated with benzo[a]pyrene (B[a]P), N-ethyl-N-nitrosourea (ENU), ethyl methanesulphonate, and 7,12-dimethylbenz[a]anthracene, with near maximum mutant frequencies measured eight days after treatment. The CD90 deficiency in mutant cells quantified by flow cytometry was shown to be due to loss of GPI anchors in a limiting-dilution cloning assay using proaerolysin selection. Individual CD90-deficient cells from cultures treated with ENU, B[a]P, and vehicle were sorted and clonally expanded for molecular analysis of their Pig-a gene. Pig-a mutations with agent-specific signatures were found in nearly all clones that developed from sorted CD90-deficient cells. These results indicate that a Pig-a mutation assay can be successfully conducted in L5178YTk cells. The assay may be useful for mutagenicity screening of environmental agents as well as for testing hypotheses in vitro before committing to in vivo Pig-a assays. Environ. Mol. Mutagen. 59:4-17, 2018. Published 2017. This article is a US Government work and is in the public domain in the USA.
X连锁的Pig-a基因编码糖基磷脂酰肌醇（GPI）锚生物合成所需的一种酶。Pig-a突变细胞无法合成GPI，也无法在其表面表达GPI锚定蛋白标志物（如CD90）。在各种体内试验中，标志物缺陷可作为Pig-a突变的表型指标。在此，我们描述了一种在L5178YTk小鼠淋巴瘤细胞中进行的体外Pig-a突变试验，其中通过使用荧光抗CD90抗体的流式细胞术来检测突变表型细胞。在用苯并[a]芘（B[a]P）、N-乙基-N-亚硝基脲（ENU）、甲磺酸乙酯和7,12-二甲基苯并[a]蒽处理的细胞中，检测到CD90缺陷突变体的频率增加，处理后八天测得接近最大突变频率。在使用气单胞菌溶素选择的有限稀释克隆试验中，通过流式细胞术定量的突变细胞中的CD90缺陷被证明是由于GPI锚的丢失。对用ENU、B[a]P和溶剂处理的培养物中的单个CD90缺陷细胞进行分选并克隆扩增，以对其Pig-a基因进行分子分析。在从分选的CD90缺陷细胞发育而来的几乎所有克隆中都发现了具有试剂特异性特征的Pig-a突变。这些结果表明，Pig-a突变试验可以在L5178YTk细胞中成功进行。该试验可能有助于环境因子的致突变性筛选，以及在进行体内Pig-a试验之前在体外检验假设。《环境与分子突变》59:4 - 17，2018年。2017年发表。本文是美国政府作品，在美国属于公共领域。
