Development of a novel genotoxicity evaluation method using a next-generation sequencer, linking chemical-induced mutations to human cancer

Abstract To better understand the cancer risk posed by radiation and the development of radiation therapy resistant cancer cells, we investigated the involvement of the cancer risk factor, APOBEC3B, in the generation of radiation-induced mutations. Expression of APOBEC3B in response to irradiation was determined in three human cancer cell lines by real-time quantitative PCR. Using the hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutation assay, mutations in the HPRT gene caused by irradiation were compared between APOBEC3B-deficient human hepatocellular carcinoma (HepG2) cells [APOBEC3B knocked out (KO) using CRISPR-Cas9 genome editing] and the parent cell line. Then, HPRT-mutated cells were individually cultured to perform PCR and DNA sequencing of HPRT exons. X-Irradiation induced APOBEC3B expression in HepG2, human cervical cancer epithelial carcinoma (HeLa) and human oral squamous cell carcinoma (SAS) cells. Forced expression of APOBEC3B increased spontaneous mutations. By contrast, APOBEC3B KO not only decreased the spontaneous mutation rate, but also strongly suppressed the increase in mutation frequency after irradiation in the parent cell line. Although forced expression of APOBEC3B in the nucleus caused DNA damage, higher levels of APOBEC3B tended to reduce APOBEC3B-induced γ-H2AX foci formation (a measure of DNA damage repair). Further, the number of γ-H2AX foci in cells stably expressing APOBEC3B was not much higher than that in controls before and after irradiation, suggesting that a DNA repair pathway may be activated. This study demonstrates that irradiation induces sustained expression of APOBEC3B in HepG2, HeLa and SAS cells, and that APOBEC3B enhances radiation-induced partial deletions.

Download Full-text

Chemically induced mutations in a MutaMouse reporter gene inform mechanisms underlying human cancer mutational signatures

Communications Biology ◽

10.1038/s42003-020-01174-y ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 2

Author(s):

Marc A. Beal ◽

Matthew J. Meier ◽

Danielle P. LeBlanc ◽

Clotilde Maurice ◽

Jason M. O’Brien ◽

...

Keyword(s):

Human Cancer ◽

Published Data ◽

Induced Mutations ◽

Lung Cancers ◽

Human Cancers ◽

Chemically Induced ◽

Mutation Pattern ◽

Environmental Causes ◽

Next Generation Sequencing Ngs

AbstractTransgenic rodent (TGR) models use bacterial reporter genes to quantify in vivo mutagenesis. Pairing TGR assays with next-generation sequencing (NGS) enables comprehensive mutation pattern analysis to inform mutational mechanisms. We used this approach to identify 2751 independent lacZ mutations in the bone marrow of MutaMouse animals exposed to four chemical mutagens: benzo[a]pyrene, N-ethyl-N-nitrosourea, procarbazine, and triethylenemelamine. We also collected published data for 706 lacZ mutations from eight additional environmental mutagens. We report that lacZ gene sequencing generates chemical-specific mutation signatures observed in human cancers with established environmental causes. For example, the mutation signature of benzo[a]pyrene, a carcinogen present in tobacco smoke, matched the signature associated with tobacco-induced lung cancers. Our results suggest that the analysis of chemically induced mutations in the lacZ gene shortly after exposure provides an effective approach to characterize human-relevant mechanisms of carcinogenesis and propose novel environmental causes of mutation signatures observed in human cancers.

Download Full-text

Discovery of mutations in Chenopodium quinoa Willd through EMS mutagenesis and mutation screening using pre-selection phenotypic data and next-generation sequencing

The Journal of Agricultural Science ◽

10.1017/s0021859619000182 ◽

2018 ◽

Vol 156 (10) ◽

pp. 1196-1204 ◽

Cited By ~ 1

Author(s):

Camilo Mestanza ◽

Ricardo Riegel ◽

Santiago C. Vásquez ◽

Diana Veliz ◽

Nicolás Cruz-Rosero ◽

...

Keyword(s):

Next Generation Sequencing ◽

Mutation Screening ◽

Chenopodium Quinoa ◽

Acetolactate Synthase ◽

Screening Strategy ◽

Next Generation ◽

Induced Mutations ◽

Phenotypic Data ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

AbstractQuinoa (Chenopodium quinoaWilld) is a dicotyledonous annual species belonging to the family Amaranthaceae, which is nutritionally well balanced in terms of its oil, protein and carbohydrate content. Targeting-induced local lesions in genomes (the TILLING strategy) was employed to find mutations in acetolactate synthase (AHAS) genes in a mutant quinoa population. TheAHASgenes were targeted because they are common enzyme target sites for five herbicide groups. Ethyl methane sulfonate (EMS) was used to induce mutations in theAHASgenes; it was found that 2% EMS allowed a mutation frequency of one mutation every 203 kilobases to be established. In the mutant population created, a screening strategy using pre-selection phenotypic data and next-generation sequencing (NGS) allowed identification of a mutation that alters the amino acid composition of this species (nucleotide 1231 codon GTT→ATT, Val→Ile); however, this mutation did not result in herbicide resistance. The current work shows that TILLING combined with the high-throughput of NGS technologies and an overlapping pool design provides an efficient and economical method for detecting induced mutations in pools of individuals.

Download Full-text