CC BY 4.0 · Pharmaceutical Fronts 2021; 03(02): e77-e85
DOI: 10.1055/s-0041-1735146
Original Article

In Vitro PIG-A Gene Mutation Assay in Human B-Lymphoblastoid TK6 Cells

Chang-Hui Zhou#
1   Shanghai Innostar Bio-tech Co., Ltd., China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China
,
Chun-Rong Yu#
1   Shanghai Innostar Bio-tech Co., Ltd., China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China
,
Peng-Cheng Huang
1   Shanghai Innostar Bio-tech Co., Ltd., China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China
,
Ruo-Wan Li
1   Shanghai Innostar Bio-tech Co., Ltd., China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China
,
Jing-Ting Wang
1   Shanghai Innostar Bio-tech Co., Ltd., China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China
,
Tian-Tian Zhao
1   Shanghai Innostar Bio-tech Co., Ltd., China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China
,
Ze-Hao Zhao
1   Shanghai Innostar Bio-tech Co., Ltd., China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China
,
Jing Ma
1   Shanghai Innostar Bio-tech Co., Ltd., China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China
,
Yan Chang
1   Shanghai Innostar Bio-tech Co., Ltd., China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China
› Author Affiliations
Funding This study was financially supported by the Major Projects Foundation of the National Health Commission of the People's Republic of China (Grant No. 2018ZX09201017-008).

Abstract

The X-linked PIG-A gene is involved in the biosynthesis of glycosylphosphatidylinositol (GPI) anchors. PIG-A mutant cells fail to synthesize GPI and to express GPI-anchored protein markers (e.g., CD59 and CD55). In recent years, in vitro PIG-A assay has been established based on the high conservation of PIG-A/Pig-a loci among different species and the large data from the in vivo system. The purpose of this study was to extend the approach for PIG-A mutation assessment to in vitro human B-lymphoblastoid TK6 cells by detecting the loss of GPI-linked CD55 and CD59 proteins. TK6 cells were treated with three mutagens 7,12-dimethylbenz[a]anthracene (DMBA), N-ethyl-N-nitrosourea (ENU), etoposide (ETO), and two nonmutagens: cadmium chloride (CdCl2) and sodium chloride (NaCl). The mutation rate of PIG-A gene within TK6 cells was determined on the 11th day with flow cytometry analysis for the negative frequencies of CD55 and CD59. The antibodies used in this production were APC mouse-anti-human CD19 antibody, PE mouse anti-human CD55 antibody, PE mouse anti-human CD59 antibody, and nucleic acid dye 7-AAD. An immunolabeling method was used to reduce the high spontaneous level of preexisting PIG-A mutant cells. Our data suggested that DMBA-, ENU-, and ETO-induced mutation frequency of PIG-A gene was increased by twofold compared with the negative control, and the effects were dose-dependent. However, CdCl2 and NaCl did not significantly increase the mutation frequency of PIG-A gene, with a high cytotoxicity at a dose of 10 mmol/L. Our study suggested that the novel in vitro PIG-A gene mutation assay within TK6 cells may represent a complement of the present in vivo Pig-a assay, and may provide guidance for their potential use in genotoxicity even in cells with a significant deficiency of GPI anchor.

# These authors contributed equally to this work.




Publication History

Received: 14 April 2021

Accepted: 24 June 2021

Article published online:
02 September 2021

© 2021. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/)

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